阅读说明：本技术 脂蛋白a亚型kiv-2结构域的拷贝数量检测试剂盒 (Copy number detection kit for lipoprotein A subtype KIV-2 structural domain ) 是由 沈林 李文涛 高鹏飞 杨敬敏 何方 郭志晨 张贝 于 2020-08-17 设计创作，主要内容包括：本发明涉及生物技术领域,特别涉及一种从基因水平检测Apo A分子KIV-2亚型结构域的重复数检测的试剂盒及其检测方法。本发明的试剂盒采用基因合成的方法合成KIV-2已知重复数的标准品,解决了无阳性样本或者样本样本量有限的确定；采用SYBR Green qPCR检测方法,能够高灵敏和特异性的检测总量≥1ng的基因组DNA,在1-2内天快速得到结果。(The invention relates to the technical field of biology, in particular to a kit for detecting the repeat number of an Apo A molecule KIV-2 subtype structural domain from a gene level and a detection method thereof. The kit adopts a gene synthesis method to synthesize a standard substance with known repeat number of KIV-2, thereby solving the problem of determination of non-positive samples or limited sample amount; by adopting an SYBR Green qPCR detection method, the genomic DNA with the total amount of more than or equal to 1ng can be detected with high sensitivity and specificity, and the result can be quickly obtained within 1-2 days.)

1. The primer combination is characterized by comprising a first combination and a second combination:

the combination is as follows:

(1) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and

(2) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; or

(3) A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (1) or (2), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (1) or (2); or

(4) A nucleotide sequence with at least 80% of identity with the nucleotide sequence shown in (1) or (2);

combining two:

(5) the upstream primer has a nucleotide sequence shown as SEQ ID No. 3; and

(6) the downstream primer has a nucleotide sequence shown as SEQ ID No. 4; or

(7) A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (5) or (6), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (5) or (6); or

(8) And (3) a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in (5) or (6).

2. The primer combination of claim 1, wherein the plurality of one or more substitutions, deletions or additions is 2, 3, 4, 5, 6, 7, 8, 9, 10.

3. Use of a primer combination according to claim 1 or 2 for the preparation of a reagent and/or a kit for detecting the number of repeats of a KIV-2 domain.

4. Use of a primer combination according to claim 1 or 2 for the preparation of a reagent and/or a kit for the detection of arteriosclerotic cardiovascular and cerebrovascular diseases.

5. A reagent for detecting the number of repeats of KIV-2 domain or for detecting arteriosclerotic cardiovascular and cerebrovascular diseases, comprising the primer set according to claim 1 or 2.

6. Kit for detecting the number of repeated KIV-2 domains or for detecting arteriosclerotic cardiovascular and cerebrovascular diseases, comprising the primer combination according to claim 1 or 2 and a conventional auxiliary agent or carrier.

7. The kit of claim 5, further comprising a standard having the sequence:

(1) has a nucleotide sequence shown as SEQ ID No. 5; or

(2) A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (1), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (1); or

(3) And (2) a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in (1).

A method for detecting the number of repeated KIV-2 domains, which comprises mixing a sample to be detected with the primer combination of claim 1 or 2, the reagent of claim 5 or the primer combination of the kit of any one of claims 6 to 7, amplifying, and detecting.

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting the repeat number of an Apo A molecule KIV-2 subtype structural domain from a gene level and a detection method thereof.

Background

Lipoprotein a (lp (a)) is a special lipoprotein particle, and the concentration of serum lp (a) is mainly controlled by gene, and can be used as an independent risk factor index of arteriosclerotic cardiovascular and cerebrovascular diseases. Its structure is very similar to LDL in protein but with an apolipoprotein apo (a) protein rich in carbohydrates and highly hydrophilic. The number of kringle4-2 domain repeats in apo (a) proteins is determined by the gene, and the number of KIV-2 type repeats per apo (a) particle varies from 3 to 40, so that the size of apo (a) is highly variable not only from individual to individual but also from one individual to another.

The correlation between kringle4-2 type repeat number, lp (a) concentration and disease was investigated. In the existing method, a known KIV-2 type repetition number sample and a KIV-2 type repetition number of a sample to be detected are detected mainly through real-time fluorescent quantitative PCR detection, and the KIV-2 type repetition number of the sample to be detected is calculated. The method needs to detect the KIV-2 type repeat number, so that no positive sample exists, the related experiment cannot be carried out, and the quantity of the positive sample cannot be realized in mass production.

Therefore, it is of great practical significance to provide a kit for detecting the number of repeats of the KIV-2 subtype domain of Apo A molecules from the gene level and a detection method thereof.

Disclosure of Invention

In view of the above, the present invention provides a diagnostic kit for detecting the number of repeats of KIV-2 domain, a preparation method thereof, and a detection method thereof. Through gene synthesis of the standard substance, the problem that the standard substance has larger difference is solved, and the accuracy of the detection method is improved; the sensitivity and specificity of detection are improved by qPCR and design of specific primers.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a primer combination, which is characterized by comprising a first combination and a second combination:

the combination is as follows:

(1) the upstream primer has a nucleotide sequence shown as SEQ ID No. 1; and

(2) the downstream primer has a nucleotide sequence shown as SEQ ID No. 2; or

(3) A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (1) or (2), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (1) or (2); or

(4) A nucleotide sequence with at least 80% of identity with the nucleotide sequence shown in (1) or (2);

combining two:

(5) the upstream primer has a nucleotide sequence shown as SEQ ID No. 3; and

(6) the downstream primer has a nucleotide sequence shown as SEQ ID No. 4; or

(7) A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (5) or (6), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (5) or (6); or

(8) And (3) a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in (5) or (6).

In some embodiments of the invention, more than one of said one or more substitutions, deletions or additions is 2, 3, 4, 5, 6, 7, 8, 9, 10.

The invention also provides application of the primer combination in preparation of a reagent and/or a kit for detecting the repeated number of KIV-2 domains.

On the basis of the research, the invention also provides application of the primer combination in preparing a reagent and/or a kit for detecting arteriosclerotic cardiovascular and cerebrovascular diseases.

The invention also provides a reagent for detecting the repeated quantity of the KIV-2 structural domain or detecting the arteriosclerotic cardiovascular and cerebrovascular diseases, which comprises the primer combination.

On the basis of the research, the invention also provides a kit for detecting the repeat quantity of the KIV-2 structural domain or detecting the arteriosclerotic cardiovascular and cerebrovascular diseases, which comprises the primer combination and a commonly used auxiliary agent or carrier.

In some embodiments of the invention, the kit further comprises a standard having the sequence:

(1) has a nucleotide sequence shown as SEQ ID No. 5; or

(2) A nucleotide sequence obtained by substituting, deleting or adding one or more bases in the nucleotide sequence shown in (1), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in (1); or

(3) And (2) a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in (1).

On the basis of the research, the invention also provides a method for detecting the repeated quantity of the KIV-2 structural domain, which comprises the steps of mixing a sample to be detected with the primer combination, the reagent or the primer combination in the kit, amplifying and detecting.

On the basis of the research, the invention also provides a detection method of the arteriosclerotic cardiovascular and cerebrovascular diseases, which comprises the steps of taking a sample to be detected, mixing the sample with the primer combination, the reagent or the primer combination in the kit, amplifying and detecting.

The kit adopts a gene synthesis method to synthesize a standard substance with known repeat number of KIV-2, thereby solving the problem of determination of non-positive samples or limited sample amount; by adopting an SYBR Green qPCR detection method, the genomic DNA with the total amount of more than or equal to 1ng can be detected with high sensitivity and specificity, and the result can be quickly obtained within 1-2 days.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a graphical representation of the KIV and ALB primer dissolution curve results in the patent, and the specificity and the repeatability of the primers are verified by adopting a SYBR Green qPCR detection method;

FIG. 2 shows a graphical representation of the KIV and ALB primer dissolution curve results in the patent, and the specificity of the primers and the difference of Ct values of two pairs of primers are verified by adopting a SYBR Green qPCR detection method.

Detailed Description

The invention discloses a diagnostic kit for detecting the number of repeated KIV-2 structural domains and a detection method thereof, and a person skilled in the art can realize the detection by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The diagnostic kit for detecting the number of repeated KIV-2 domains and the detection method thereof provided by the invention can be purchased from the market.

The invention is further illustrated by the following examples: