CN111440862A

CN111440862A - Primer group, kit and method for evaluating prognosis of chronic heart failure patient

Info

Publication number: CN111440862A
Application number: CN202010424914.6A
Authority: CN
Inventors: 汪道文; 胡冬; 李诗洋
Original assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Current assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-07-24

Abstract

The invention discloses a primer group, a kit and a method for evaluating prognosis of a patient with chronic heart failure. The primer group comprises: the primer set 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, an eighth downstream primer, a ninth upstream primer, a ninth downstream primer, a tenth upstream primer, a tenth downstream primer, an eleventh upstream primer, an eleventh downstream primer, a twelfth upstream primer, a twelfth downstream primer, a thirteenth upstream primer, a thirteenth downstream primer and a fourteenth upstream primer. The primer group can accurately amplify the SNP locus in a sample to be detected, so that an amplification product can accurately reflect the prognosis condition of a patient with chronic heart failure.

Description

Primer group, kit and method for evaluating prognosis of chronic heart failure patient

Technical Field

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

Background

Chronic heart failure is the terminal stage of various cardiovascular diseases, which are affected by various environmental factors including hypertension, diabetes, hyperlipidemia, and the like. Patients with chronic heart failure manifest chronic fatigue, shortness of breath, dyspnea, pulmonary congestion and edema due to inadequate cardiac pumping. In adults in China, the prevalence rate of chronic heart failure is about 0.9%, women is higher than men, and at present, over 400 thousands of chronic heart failure patients in China become a very serious social problem. At present, although a plurality of medicines and auxiliary equipment for treating chronic heart failure appear, the prognosis of chronic heart failure patients is still not optimistic, and the 5-year survival rate is equivalent to that of malignant tumors. Therefore, it is important to perform prognosis evaluation on chronic heart failure patients at an early stage and guide intensive treatment according to the prognosis evaluation result.

The existing prognosis evaluation technology for chronic heart failure is mainly amino-terminal brain natriuretic peptide (NT-proBNP) detection. The NT-proBNP detection reflects the deterioration degree of the chronic heart failure by utilizing the level of a cardiac function marker NT-proBNP, and although the detection method is widely applied to the evaluation of chronic heart failure, the accuracy of the detection method is influenced by age and obesity degree, so that the detection result is inaccurate.

Disclosure of Invention

In order to solve the problems in the prior art, the embodiment of the invention provides a primer set, a kit and a method for evaluating prognosis of a patient with chronic heart failure. The technical scheme is as follows:

in one aspect, the present invention provides a primer set for assessing prognosis of a patient with chronic heart failure, the primer set comprising: 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, an eighth downstream primer, a ninth upstream primer, a ninth downstream primer, a tenth upstream primer, a tenth downstream primer, an eleventh upstream primer, an eleventh downstream primer, a twelfth upstream primer, a twelfth downstream primer, a thirteenth upstream primer, a thirteenth downstream primer, a fourteenth upstream primer, a fourteenth downstream primer, a fifteenth upstream primer, a fifteenth downstream primer, a sixteenth upstream primer, a sixteenth downstream primer, a seventeenth upstream primer, a seventeenth downstream primer, a seventh downstream primer, a primer, an eighteenth upstream primer, an eighteenth downstream primer, a nineteenth upstream primer, a nineteenth downstream primer, a twentieth upstream primer, a twentieth downstream primer, a twenty-first upstream primer, a twenty-first downstream primer, a twenty-second upstream primer, a twenty-second downstream primer, a twenty-third upstream primer, a twenty-third downstream primer, a twenty-fourth upstream primer, a twenty-fourth downstream primer, a twenty-fifth upstream primer, a twenty-fifth downstream primer, a twenty-sixth upstream primer, a twenty-sixth downstream primer, a twenty-seventh upstream primer, a twenty-seventh downstream primer, a twenty-eighth upstream primer, a twenty-eighth downstream primer, a twenty-ninth upstream primer, a twenty-ninth downstream primer, a thirty-upstream primer, a thirty-downstream primer, a thirty-eleventh upstream primer and a thirty-eleventh downstream primer.

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 in the sequence table, the sequence of the fourth downstream primer is shown as SEQ ID NO. 8 in the sequence table, the sequence of the fifth upstream primer is shown as SEQ ID NO. 9 in the sequence table, and the sequence of the fifth downstream primer is shown as SEQ ID NO. 10 in the sequence table, 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: as shown at 16, the flow of the gas, the sequence of the ninth upstream primer is shown as SEQ ID NO: as shown in (17) of the drawings, the sequence of the ninth downstream primer is shown as SEQ ID NO: as shown at 18, the flow of air is, the sequence of the tenth upstream primer is shown as SEQ ID NO: as indicated at 19, the flow of air is, the sequence of the tenth downstream primer is shown as SEQ ID NO: shown at 20.

The sequence of the eleventh upstream primer is shown as SEQ ID NO:21 in the sequence table, the sequence of the eleventh downstream primer is shown as SEQ ID NO:22 in the sequence table, the sequence of the twelfth upstream primer is shown as SEQ ID NO:23 in the sequence table, the sequence of the twelfth downstream primer is shown as SEQ ID NO:24 in the sequence table, the sequence of the thirteenth upstream primer is shown as SEQ ID NO:25 in the sequence table, the sequence of the thirteenth downstream primer is shown as SEQ ID NO:26 in the sequence table, the sequence of the fourteenth upstream primer is shown as SEQ ID NO:27 in the sequence table, the sequence of the fourteenth downstream primer is shown as SEQ ID NO:28 in the sequence table, the sequence of the fifteenth upstream primer is shown as SEQ ID NO:29 in the sequence table, the sequence of the fifteenth downstream primer is shown as SEQ ID NO:30 in the sequence table, the sequence of the sixteenth upstream primer is shown as SEQ ID NO. 31 in the sequence table, the sequence of the sixteenth downstream primer is shown as SEQ ID NO. 32 in the sequence table, the sequence of the seventeenth upstream primer is shown as SEQ ID NO. 33 in the sequence table, the sequence of the seventeenth downstream primer is shown as SEQ ID NO. 34 in the sequence table, the sequence of the eighteenth upstream primer is shown as SEQ ID NO. 35 in the sequence table, the sequence of the eighteenth downstream primer is shown as SEQ ID NO. 36 in the sequence table, the sequence of the nineteenth upstream primer is shown as SEQ ID NO. 37 in the sequence table, the sequence of the nineteenth downstream primer is shown as SEQ ID NO. 38 in the sequence table, the sequence of the twentieth upstream primer is shown as SEQ ID NO. 39 in the sequence table, and the sequence of the twentieth downstream primer is shown as SEQ ID NO. 40 in the sequence table,

the sequence of the twenty-first upstream primer is shown as SEQ ID NO 41 in the sequence table, the sequence of the twenty-first downstream primer is shown as SEQ ID NO 42 in the sequence table, the sequence of the twenty-second upstream primer is shown as SEQ ID NO 43 in the sequence table, the sequence of the twenty-second downstream primer is shown as SEQ ID NO 44 in the sequence table, the sequence of the twenty-third upstream primer is shown as SEQ ID NO 45 in the sequence table, the sequence of the twenty-third downstream primer is shown as SEQ ID NO 46 in the sequence table, the sequence of the twenty-fourth upstream primer is shown as SEQ ID NO 47 in the sequence table, the sequence of the twenty-fourth downstream primer is shown as SEQ ID NO 48 in the sequence table, the sequence of the twenty-fifth upstream primer is shown as SEQ ID NO 49 in the sequence table, and the sequence of the twenty-fifth downstream primer is shown as SEQ ID NO 50 in the sequence table, the sequence of the twenty-sixth upstream primer is shown as SEQ ID NO 51 in the sequence table, the sequence of the twenty-sixth downstream primer is shown as SEQ ID NO 52 in the sequence table, the sequence of the twenty-seventh upstream primer is shown as SEQ ID NO 53 in the sequence table, the sequence of the twenty-seventh downstream primer is shown as SEQ ID NO 54 in the sequence table, the sequence of the twenty-eighth upstream primer is shown as SEQ ID NO 55 in the sequence table, the sequence of the twenty-eighth downstream primer is shown as SEQ ID NO 56 in the sequence table, the sequence of the twenty-ninth upstream primer is shown as SEQ ID NO 57 in the sequence table, the sequence of the twenty-ninth downstream primer is shown as SEQ ID NO 58 in the sequence table, the sequence of the thirty-upstream primer is shown as SEQ ID NO 59 in the sequence table, and the sequence of the thirty-downstream primer is shown as SEQ ID NO 60 in the sequence table, the sequence of the thirty-first upstream primer is shown as SEQ ID NO. 61 in the sequence table, and the sequence of the thirty-first downstream primer is shown as SEQ ID NO. 62 in the sequence table.

In another aspect, the embodiments of the present invention provide a kit for evaluating prognosis of a patient with chronic heart failure, the kit comprising the primer set according to claim 1.

Specifically, the kit also comprises TaKaRa Taq HS, 10 × PCR Buffer, dNTP mix, Primer Pool 1/2 and sterilized water.

In yet another aspect, the embodiments of the present invention provide a method for assessing prognosis of a patient with chronic heart failure using the primer set as described above, the method comprising:

the method comprises the steps of extracting genome DNA of a sample to be detected, amplifying the genome DNA through a primer group to obtain 31 amplification products, purifying the 31 amplification products respectively to obtain 31 purified products, sequencing the 31 purified products for one generation to obtain 31 sequencing products, assigning each sequencing product, assigning a value to be 1 when an SNP site corresponding to the sequencing product is a protection factor, assigning a value to be 1 × HR when the SNP site corresponding to the sequencing product is a risk factor, assigning a value to be 1 HR and HR to be a risk ratio, adding the 31 sequencing products after assigning, wherein the sum is X, the sample to be detected is at low risk if X is 34.82-38.20, the sample to be detected is at medium risk if X is 38.20-39.26, and the sample to be detected is at high risk if X is 39.26-42.23.

Specifically, the procedure for amplification comprises, for each cycle: at 98 ℃ for 10s, 55 ℃ for 30s and 72 ℃ for 1min for 35 cycles.

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 provided by the invention respectively correspond to 31 risk alleles with sensitive prognosis, so that the accuracy and comprehensiveness of a prognosis evaluation result can be ensured, meanwhile, the length of an amplification product obtained by the primer group is 300-500 bp, and an SNP locus is designed in the middle position of the amplification product, so that the 5 'end interference result and the 3' end passivation phenomenon can be effectively prevented, the primer group can accurately amplify the SNP locus in a sample to be detected, so that the amplification product can accurately reflect the prognosis condition of a patient with chronic heart failure, 31 amplification primers can be used for simultaneously sequencing in the same PCR amplification mode, and the sequencing accuracy is further ensured. The method provided by the invention can accurately identify the gene mutation related to prognosis by sequencing and data interpretation of a target amplification region, thereby accurately evaluating the prognosis and providing a timely and reliable detection report for clinic, meanwhile, the method can reach 100% in accuracy by one generation of sequencing and typing point mutation, and has the characteristics of different results due to different primary factors because of obvious heterogeneity of chronic heart failure.

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 diagram of sequencing peaks corresponding to amplification products obtained by amplification with a first forward primer and a first downstream primer provided in the third embodiment of the present invention;

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

FIG. 3 is a diagram of sequencing peaks corresponding to amplification products obtained by amplification with a third forward primer and a third downstream primer provided in the third embodiment of the present invention;

FIG. 4 is a diagram of sequencing peaks corresponding to amplification products obtained by amplification with the fourth forward primer and the fourth downstream primer provided in the third embodiment of the present invention;

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

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

FIG. 7 is a sequencing peak diagram of an amplification product obtained by amplification with the seventh forward primer and the seventh downstream primer provided in the third embodiment of the present invention;

FIG. 8 is a sequencing peak diagram of an amplification product obtained by amplifying the eighth forward primer and the eighth backward primer provided in the third embodiment of the present invention;

FIG. 9 is a sequencing peak diagram of an amplification product obtained by amplification of the ninth forward primer and the ninth backward primer provided in the third embodiment of the present invention;

FIG. 10 is a diagram of sequencing peaks corresponding to amplification products obtained by amplification with the tenth forward primer and the tenth backward primer provided in the third embodiment of the present invention;

FIG. 11 is a sequencing peak diagram of an amplification product obtained by amplifying the eleventh forward primer and the eleventh reverse primer provided in the third embodiment of the present invention;

FIG. 12 is a sequencing peak diagram of an amplification product obtained by amplification of a twelfth forward primer and a twelfth downstream primer provided in the third embodiment of the present invention;

FIG. 13 is a sequencing peak diagram of an amplification product obtained by amplifying a thirteenth upstream primer and a thirteenth downstream primer provided in the third embodiment of the present invention;

FIG. 14 is a sequencing peak diagram of an amplification product obtained by amplifying a fourteenth forward primer and a fourteenth backward primer provided in the third embodiment of the present invention;

FIG. 15 is a sequencing peak diagram of an amplification product obtained by amplifying a fifteenth forward primer and a fifteenth backward primer provided in the third embodiment of the present invention;

FIG. 16 is a sequencing peak diagram of an amplification product obtained by amplifying the sixteenth upstream primer and the sixteenth downstream primer provided in the third embodiment of the present invention;

FIG. 17 is a sequencing peak diagram of an amplification product obtained by amplifying the seventeenth forward primer and the seventeenth backward primer provided in the third embodiment of the present invention;

FIG. 18 is a sequencing peak diagram of an amplification product obtained by amplifying the eighteenth forward primer and the eighteenth backward primer provided in the third embodiment of the present invention;

FIG. 19 is a sequencing peak diagram of an amplification product obtained by amplifying a nineteenth forward primer and a nineteenth backward primer provided in the third embodiment of the present invention;

FIG. 20 is a sequencing peak diagram of the amplification product obtained by the amplification of the twentieth upstream primer and the twentieth downstream primer provided in the third embodiment of the present invention;

FIG. 21 is a diagram showing sequencing peaks corresponding to amplification products obtained by amplifying the twenty-first upstream primer and the twenty-first downstream primer provided in the third embodiment of the present invention;

FIG. 22 is a sequencing peak diagram of an amplification product obtained by amplifying the twenty-second forward primer and the twenty-second backward primer provided in the third embodiment of the present invention;

FIG. 23 is a sequencing peak diagram of an amplification product obtained by amplifying the twenty-third forward primer and the twenty-third backward primer provided in the third embodiment of the present invention;

FIG. 24 is a sequencing peak diagram of an amplification product obtained by amplifying a twenty-fourth forward primer and a twenty-fourth reverse primer provided in the third embodiment of the present invention;

FIG. 25 is a diagram showing sequencing peaks corresponding to amplification products obtained by amplifying the twenty-fifth upstream primer and the twenty-fifth downstream primer provided in the third embodiment of the present invention;

FIG. 26 is a diagram showing sequencing peaks corresponding to amplification products obtained by amplifying a twenty-sixth upstream primer and a twenty-sixth downstream primer provided in the third embodiment of the present invention;

FIG. 27 is a diagram showing sequencing peaks corresponding to amplification products obtained by amplifying the twenty-seventh upstream primer and the twenty-seventh downstream primer provided in the third embodiment of the present invention;

FIG. 28 is a sequencing peak diagram of an amplification product obtained by amplifying the twenty-eighth forward primer and the twenty-eighth reverse primer provided in the third embodiment of the present invention;

FIG. 29 is a sequencing peak diagram of an amplification product obtained by amplifying a twenty-ninth upstream primer and a twenty-ninth downstream primer provided in the third embodiment of the present invention;

FIG. 30 is a sequencing peak diagram corresponding to an amplification product obtained by amplifying a thirtieth upstream primer and a thirtieth downstream primer provided in the third embodiment of the present invention;

FIG. 31 is a sequencing peak diagram of an amplification product obtained by amplifying the thirty-first upstream primer and the thirty-first downstream 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 are described in further detail below.

Example one

The embodiment of the invention provides a primer group for evaluating prognosis of a patient with chronic heart failure, which comprises the following components: 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, an eighth downstream primer, a ninth upstream primer, a ninth downstream primer, a tenth upstream primer, a tenth downstream primer, an eleventh upstream primer, an eleventh downstream primer, a twelfth upstream primer, a twelfth downstream primer, a thirteenth upstream primer, a thirteenth downstream primer, a fourteenth upstream primer, a fourteenth downstream primer, a fifteenth upstream primer, a fifteenth downstream primer, a sixteenth upstream primer, a sixteenth downstream primer, a seventeenth upstream primer, a seventeenth downstream primer, an eighteenth upstream primer, a sixteenth upstream primer, a seventeenth upstream primer, a seventh upstream primer, a, An eighteenth downstream primer, a nineteenth upstream primer, a nineteenth downstream primer, a twentieth upstream primer, a twentieth downstream primer, a twenty-first upstream primer, a twenty-first downstream primer, a twenty-second upstream primer, a twenty-second downstream primer, a twenty-third upstream primer, a twenty-third downstream primer, a twenty-fourth upstream primer, a twenty-fourth downstream primer, a twenty-fifth upstream primer, a twenty-fifth downstream primer, a twenty-sixth upstream primer, a twenty-sixth downstream primer, a twenty-seventh upstream primer, a twenty-seventh downstream primer, a twenty-eighth upstream primer, a twenty-eighth downstream primer, a twenty-ninth upstream primer, a twenty-ninth downstream primer, a thirty-upstream primer, a thirty-downstream primer, a thirty-eleventh upstream primer and a thirty-eleventh downstream primer.

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 in the sequence table, the sequence of the fourth downstream primer is shown as SEQ ID NO. 8 in the sequence table, the sequence of the fifth upstream primer is shown as SEQ ID NO. 9 in the sequence table, the sequence of the fifth downstream primer is shown as SEQ ID NO. 10 in the sequence table, and the sequence of the sixth upstream primer is shown as SEQ ID NO. 11 in the sequence table, the sequence of the sixth downstream primer is shown as SEQ ID NO. 12 in the sequence table, the sequence of the seventh upstream primer is shown as SEQ ID NO. 13 in the sequence table, the sequence of the seventh downstream primer is shown as SEQ ID NO. 14 in the sequence table, the sequence of the eighth upstream primer is shown as SEQ ID NO. 15 in the sequence table, the sequence of the eighth downstream primer is shown as SEQ ID NO. 16 in the sequence table, the sequence of the ninth upstream primer is shown as SEQ ID NO. 17 in the sequence table, the sequence of the ninth downstream primer is shown as SEQ ID NO. 18 in the sequence table, the sequence of the tenth upstream primer is shown as SEQ ID NO. 19 in the sequence table, and the sequence of the tenth downstream primer is shown as SEQ ID NO. 20 in the sequence table,

the sequence of the eleventh upstream primer is shown as SEQ ID NO. 21 in the sequence table, the sequence of the eleventh downstream primer is shown as SEQ ID NO. 22 in the sequence table, the sequence of the twelfth upstream primer is shown as SEQ ID NO. 23 in the sequence table, the sequence of the twelfth downstream primer is shown as SEQ ID NO. 24 in the sequence table, the sequence of the thirteenth upstream primer is shown as SEQ ID NO. 25 in the sequence table, the sequence of the thirteenth downstream primer is shown as SEQ ID NO. 26 in the sequence table, the sequence of the fourteenth upstream primer is shown as SEQ ID NO. 27 in the sequence table, the sequence of the fourteenth downstream primer is shown as SEQ ID NO. 28 in the sequence table, the sequence of the fifteenth upstream primer is shown as SEQ ID NO. 29 in the sequence table, the sequence of the fifteenth downstream primer is shown as SEQ ID NO. 30 in the sequence table, the sequence of the sixteenth upstream primer is shown as SEQ ID NO. 31 in the sequence table, the sequence of the sixteenth downstream primer is shown as SEQ ID NO. 32 in the sequence table, the sequence of the seventeenth upstream primer is shown as SEQ ID NO. 33 in the sequence table, the sequence of the seventeenth downstream primer is shown as SEQ ID NO. 34 in the sequence table, the sequence of the eighteenth upstream primer is shown as SEQ ID NO. 35 in the sequence table, the sequence of the eighteenth downstream primer is shown as SEQ ID NO. 36 in the sequence table, the sequence of the nineteenth upstream primer is shown as SEQ ID NO. 37 in the sequence table, the sequence of the nineteenth downstream primer is shown as SEQ ID NO. 38 in the sequence table, the sequence of the twentieth upstream primer is shown as SEQ ID NO. 39 in the sequence table, and the sequence of the twentieth downstream primer is shown as SEQ ID NO. 40 in the sequence table.

The sequence of the twenty-first upstream primer is shown as SEQ ID NO 41 in the sequence table, the sequence of the twenty-first downstream primer is shown as SEQ ID NO 42 in the sequence table, the sequence of the twenty-second upstream primer is shown as SEQ ID NO 43 in the sequence table, the sequence of the twenty-second downstream primer is shown as SEQ ID NO 44 in the sequence table, the sequence of the twenty-third upstream primer is shown as SEQ ID NO 45 in the sequence table, the sequence of the twenty-third downstream primer is shown as SEQ ID NO 46 in the sequence table, the sequence of the twenty-fourth upstream primer is shown as SEQ ID NO 47 in the sequence table, the sequence of the twenty-fourth downstream primer is shown as SEQ ID NO 48 in the sequence table, the sequence of the twenty-fifth upstream primer is shown as SEQ ID NO 49 in the sequence table, the sequence of the twenty-fifth downstream primer is shown as SEQ ID NO 50 in the sequence table, the sequence of the twenty-sixth upstream primer is shown as SEQ ID NO 51 in the sequence table, the sequence of the twenty-sixth downstream primer is shown as SEQ ID NO 52 in the sequence table, the sequence of the twenty-seventh upstream primer is shown as SEQ ID NO 53 in the sequence table, the sequence of the twenty-seventh downstream primer is shown as SEQ ID NO 54 in the sequence table, the sequence of the twenty-eighth upstream primer is shown as SEQ ID NO 55 in the sequence table, the sequence of the twenty-eighth downstream primer is shown as SEQ ID NO 56 in the sequence table, the sequence of the twenty-ninth upstream primer is shown as SEQ ID NO 57 in the sequence table, the sequence of the twenty-ninth downstream primer is shown as SEQ ID NO 58 in the sequence table, the sequence of the thirty-upstream primer is shown as SEQ ID NO 59 in the sequence table, the sequence of the thirty-downstream primer is shown as SEQ ID NO 60 in the sequence table, the sequence of the thirty-first upstream primer is shown as SEQ ID NO. 61 in the sequence table, and the sequence of the thirty-first downstream primer is shown as SEQ ID NO. 62 in the sequence table.

Table 1 shows the genes, SNP sites, risk genotypes and risk ratios corresponding to the primer sets provided in example one

The primer set provided by the embodiment of the invention can amplify 31 risk alleles sensitive to corresponding prognosis in a sample to be detected respectively and accurately, specifically amplify rs SNP locus corresponding to MNS gene, rs11083543 corresponding to FCGBP gene, rs SNP locus, rs61761894 corresponding to SFRP gene, rs SNP locus corresponding to C16orf gene, rs420137, rs638551, rs653521 SNP locus corresponding to FND gene, rs SNP locus corresponding to NUDT gene, rs SNP locus corresponding to SERPINB gene, rs 10961617 corresponding to FREM gene, rs SNP locus corresponding to RS SNP locus, G T8D gene, rs SNP locus corresponding to RX C gene, rs SNP locus corresponding to MED gene, rs locus corresponding to MATN gene, rs SNP locus corresponding to S SNP locus, rs SNP locus corresponding to S11A gene, rs SNP locus corresponding to FAM83 gene, AP locus corresponding to RX C gene, rs SNP locus corresponding to MED gene, MATN gene, and corresponding to RS SNP locus, thereby the amplification result of rs SNP locus corresponding to RTK gene amplification of RTD gene can be effectively reflected between the PCR products of rs SNP loci corresponding to RTK gene amplification sites, and RK PCR products of RTK gene amplification sites of RTK gene, thereby enabling the PCR products to be accurately estimated in SNP loci corresponding to PCR products of rs SNP loci corresponding to be capable of RTK 12 SNP loci and PCR products of RTK gene amplification sites, and PCR products of RTK gene amplification genes of RTM gene amplification genes, thereby enabling the SNP loci to be capable of PCR products to be capable of accurately and capable of PCR products of accurately and accurately reflecting the SNP loci corresponding to be capable of PCR products of the SNP loci corresponding to be estimated at the SNP loci corresponding to be capable of SNP loci corresponding to be detected.

Example two

The embodiment of the invention provides a kit for evaluating prognosis of a patient with chronic heart failure, which comprises the primer group provided by the embodiment of the invention.

Specifically, the kit may further comprise TaKaRa Taq HS, 10 × PCR Buffer, dNTP mix, Primer Pool 1/2 and sterilized water.

The kit provided by the invention can respectively amplify 31 risk alleles with sensitive prognosis, which can ensure the accuracy and comprehensiveness of a prognosis evaluation result, meanwhile, the length of an amplification product obtained by the primer group is 300-500 bp, and SNP loci are designed in the middle position of the amplification product, which can effectively prevent a 5 'end interference result and a passivation phenomenon of a 3' end, so that the primer group can accurately amplify the SNP loci in a sample to be detected, thereby the amplification product can accurately reflect the prognosis condition of a patient with chronic heart failure, 31 amplification primers can simultaneously perform sequencing in the same PCR amplification mode, and the sequencing accuracy is further ensured. The method provided by the invention can accurately identify the gene mutation related to prognosis by sequencing and data interpretation of a target amplification region, thereby accurately evaluating the prognosis and providing a timely and reliable detection report for clinic, meanwhile, the method can reach 100% in accuracy by one generation of sequencing and typing point mutation, and has the characteristics of different results due to different primary factors because chronic heart failure has obvious heterogeneity.

EXAMPLE III

The embodiment of the invention provides a method for evaluating prognosis of a patient with chronic heart failure by using the kit provided in the second embodiment, which comprises the following steps:

extracting the genome DNA of a sample to be detected;

zhangyi, age 64 years old, male, hospitalization for diagnosis of coronary heart disease, ischemic cardiomyopathy, hypertension, diabetes, coronary heart disease and a history of smoking, no history of hyperlipidemia and arrhythmia, no admission history of alcohol 110/85mmHg, heart rate 87 times/min, NYH heart function classification (NYHA)4 grade, fasting plasma glucose 7.65 (mmol/L), A L T306 (U/L0), AST218 (U/L1), TC 3.09 (mmol/L2), TG 1.0 (mmol/L), Cr 142 (umol/L), HD L0.54.54 (mmol/L), L D L2.19.19 (mmol/L), NT-proBNP 19 ng/L, cardiac super-examination ventricular ejection fraction 17%, left ventricular internal diameter 81cm, left internal diameter 57cm, ventricular septum 7mm, left ventricular posterior wall 8mm, patients taking Lansimen for a long period of medication, furosemidetached tablets, and color tablets for treating cardiovascular diseases.

Specifically, the sample to be tested may be a peripheral blood sample, a body fluid sample or a tissue organ sample of a patient.

In this embodiment, the sample to be tested is a peripheral blood sample of a patient, and fasting venous blood of 5m L is taken from the patient on the day of admission, is subjected to EDTA anticoagulation treatment, and is stored at 4 ℃.

The genomic DNA of the peripheral blood of the patient was extracted using a blood DNA extraction kit provided by Tiangen Biochemical technology (Beijing) Ltd and stored at-20 ℃.

Amplifying the genome DNA by the primer group provided by the first embodiment of the invention to obtain 31 amplification products;

specifically, the amplification system is shown in table 2, specifically as follows:

TABLE 2 amplification System

Respectively purifying the 31 amplification products to obtain 31 purified products;

performing first-generation sequencing on 31 purified products to obtain 31 sequencing products;

assigning a value to each sequencing product, wherein the value is 1 when the SNP locus corresponding to the sequencing product is a protective factor, and the value is 1 × HR when the SNP locus corresponding to the sequencing product is a risk factor, wherein HR is a risk ratio;

adding the 31 assigned sequencing products, wherein the sum is X, X is the Gene Risk Score (GRS), and if X is not less than 34.82 and not more than 38.20, the prognosis is better, namely the sample to be detected is at low risk; if X is more than 38.20 and less than or equal to 39.26, centering in advance, namely the sample to be detected is at medium risk; if X is more than 39.26 and less than or equal to 42.23, the prognosis is poor, and the sample to be detected is at high risk.

In this example, HR was obtained from prognostic analysis of 998 patients with chronic heart failure, i.e. using COX proportional risk model in combination with patient follow-up data and genotyping analysis, in this example each SNP has a fixed HR value; the 31 sequencing peak diagrams of the sample to be detected provided by the embodiment of the invention are respectively shown in fig. 1 and fig. 2.

In this example, rs741164, rs420137, rs10961757, rs948847, rs3817602, rs1869608, rs17221959, rs9370340, rs35179634, rs61748727, rs6227, rs2269287, rs2297224, rs3210140, rs653521 and rs10733289 are risk genotypes, i.e., the sequencing products corresponding to the SNP sites are risk factors, and when assigning values to each sequencing product, the assignment values are 1 × HR., in this example, rs1715919, rs11083543, rs 76161894, rs3738530, rs16946429, rs 316983, rs423490, rs 1802072074, rs1463725, rs 741521143, rs 1989, rs2229682, rs1351113, rs638551 and rs 16536 are protection genotypes, i.e., the sequencing products corresponding to the SNP sites are protection factors, and when assigning values to each sequencing product, the protection factors are assigned.

In this embodiment, the assignment to the sample to be tested according to the sequence of the primers in the primer set is sequentially as follows: 1, 1, 1, 1.51, 1.43, 1, 1, 1, 1.39, 1.36, 1.43, 1, 1, 1, 1, 1.4, 1.37, 1.62, 1, 1.36, 1.6, 1, 1.51, 1, 1, 1.36, 1, 1.7, 1.8, 1.57 and 1.72, and X is 39.13 after summation, i.e. falling within the median range of prognosis, indicating a moderate risk for the prognosis of the patient.

The embodiment of the invention provides a method for evaluating the prognosis of a patient with chronic heart failure, which can accurately identify the genetic locus mutation related to the prognosis by sequencing a target amplification region and judging data, thereby providing a timely and reliable detection report for clinic. The detection method provided by the embodiment of the invention can achieve 100% of accuracy through first-generation sequencing and typing point mutation, and the outcome of chronic heart failure has obvious heterogeneity, and different characteristics of different outcomes can be caused due to different primary factors.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> affiliated Tongji hospital of Tongji medical college of Huazhong university of science and technology

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

gacgacgaca gcacagaag 19

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

ctgttttcac atctctggca aatg24

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<213> Artificial Sequence (Artificial Sequence)

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caccttcctc ttggttcagc t 21

Claims

1. A primer set for assessing prognosis of a patient with chronic heart failure, wherein the primer set 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, an eighth downstream primer, a ninth upstream primer, a ninth downstream primer, a tenth upstream primer, a tenth downstream primer, an eleventh upstream primer, an eleventh downstream primer, a twelfth upstream primer, a twelfth downstream primer, a thirteenth upstream primer, a thirteenth downstream primer, a fourteenth upstream primer, a fourteenth downstream primer, a fifteenth upstream primer, a fifteenth downstream primer, a sixteenth upstream primer, a sixteenth downstream primer, a seventeenth upstream primer, a seventeenth downstream primer, an eighteenth upstream primer, a sixteenth upstream primer, a seventeenth upstream primer, a seventh upstream primer, a, An eighteenth downstream primer, a nineteenth upstream primer, a nineteenth downstream primer, a twentieth upstream primer, a twentieth downstream primer, a twenty-first upstream primer, a twenty-first downstream primer, a twenty-second upstream primer, a twenty-second downstream primer, a twenty-third upstream primer, a twenty-third downstream primer, a twenty-fourth upstream primer, a twenty-fourth downstream primer, a twenty-fifth upstream primer, a twenty-fifth downstream primer, a twenty-sixth upstream primer, a twenty-sixth downstream primer, a twenty-seventh upstream primer, a twenty-seventh downstream primer, a twenty-eighth upstream primer, a twenty-eighth downstream primer, a twenty-ninth upstream primer, a twenty-ninth downstream primer, a thirty-upstream primer, a thirty-downstream primer, a thirty-eleventh upstream primer and a thirty-eleventh downstream primer,

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 in the sequence table, the sequence of the fourth downstream primer is shown as SEQ ID NO. 8 in the sequence table, the sequence of the fifth upstream primer is shown as SEQ ID NO. 9 in the sequence table, the sequence of the fifth downstream primer is shown as SEQ ID NO. 10 in the sequence table, the sequence of the sixth upstream primer is shown as SEQ ID NO. 11 in the sequence table, the sequence of the sixth downstream primer is shown as SEQ ID NO. 12 in the sequence table, the sequence of the seventh upstream primer is shown as SEQ ID NO. 13 in the sequence table, the sequence of the seventh downstream primer is shown as SEQ ID NO. 14 in the sequence table, the sequence of the eighth upstream primer is shown as SEQ ID NO. 15 in the sequence table, the sequence of the eighth downstream primer is shown as SEQ ID NO. 16 in the sequence table, the sequence of the ninth upstream primer is shown as SEQ ID NO. 17 in the sequence table, the sequence of the ninth downstream primer is shown as SEQ ID NO. 18 in the sequence table, the sequence of the tenth upstream primer is shown as SEQ ID NO. 19 in the sequence table, and the sequence of the tenth downstream primer is shown as SEQ ID NO. 20 in the sequence table,

the sequence of the eleventh upstream primer is shown as SEQ ID NO. 21 in the sequence table, the sequence of the eleventh downstream primer is shown as SEQ ID NO. 22 in the sequence table, the sequence of the twelfth upstream primer is shown as SEQ ID NO. 23 in the sequence table, the sequence of the twelfth downstream primer is shown as SEQ ID NO. 24 in the sequence table, the sequence of the thirteenth upstream primer is shown as SEQ ID NO. 25 in the sequence table, the sequence of the thirteenth downstream primer is shown as SEQ ID NO. 26 in the sequence table, the sequence of the fourteenth upstream primer is shown as SEQ ID NO. 27 in the sequence table, the sequence of the fourteenth downstream primer is shown as SEQ ID NO. 28 in the sequence table, the sequence of the fifteenth upstream primer is shown as SEQ ID NO. 29 in the sequence table, the sequence of the fifteenth downstream primer is shown as SEQ ID NO. 30 in the sequence table, the sequence of the sixteenth upstream primer is shown as SEQ ID NO. 31 in the sequence table, the sequence of the sixteenth downstream primer is shown as SEQ ID NO. 32 in the sequence table, the sequence of the seventeenth upstream primer is shown as SEQ ID NO. 33 in the sequence table, the sequence of the seventeenth downstream primer is shown as SEQ ID NO. 34 in the sequence table, the sequence of the eighteenth upstream primer is shown as SEQ ID NO. 35 in the sequence table, the sequence of the eighteenth downstream primer is shown as SEQ ID NO. 36 in the sequence table, the sequence of the nineteenth upstream primer is shown as SEQ ID NO. 37 in the sequence table, the sequence of the nineteenth downstream primer is shown as SEQ ID NO. 38 in the sequence table, the sequence of the twentieth upstream primer is shown as SEQ ID NO. 39 in the sequence table, and the sequence of the twentieth downstream primer is shown as SEQ ID NO. 40 in the sequence table,

2. A kit for assessing prognosis of a patient with chronic heart failure, the kit comprising the primer set according to claim 1.

3. The kit of claim 2, further comprising TaKaRa Taq HS, 10 × PCR Buffer, dNTP mix, Primer Pool 1/2 and sterilized water.

4. A method for assessing prognosis of a patient with chronic heart failure using the primer set according to claim 1, comprising:

extracting the genome DNA of a sample to be detected;

amplifying the genome DNA through the primer group to obtain 31 amplification products;

performing first-generation sequencing on the 31 purified products to obtain 31 sequencing products;

assigning a value to each sequencing product, wherein when the SNP locus corresponding to the sequencing product is a protective factor, the value is 1, and when the SNP locus corresponding to the sequencing product is a risk factor, the value is 1 × HR, and the HR is a risk ratio;

adding the 31 assigned sequencing products, wherein the sum of the addition is X, and if X is more than or equal to 34.82 and less than or equal to 38.20, the sample to be tested is at low risk; if X is greater than 38.20 and less than or equal to 39.26, the sample to be detected is at medium risk; and if X is more than 39.26 and less than or equal to 42.23, the sample to be detected is at high risk.

5. The method of claim 4, wherein the procedure for amplification comprises, for each cycle: at 98 ℃ for 10s, 55 ℃ for 30s and 72 ℃ for 1min for 35 cycles.

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