CN112391452A

CN112391452A - Kit for detection

Info

Publication number: CN112391452A
Application number: CN201910737787.2A
Authority: CN
Inventors: 张辉
Original assignee: Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
Current assignee: Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
Priority date: 2019-08-12
Filing date: 2019-08-12
Publication date: 2021-02-23

Abstract

The invention discloses a kit for detection, which is used for amplifying an IL-6 gene and sequencing an amplification product, and comprises IL-6 upstream and downstream amplification primers, DNA polymerase, an amplification buffer solution and dNTPs. The invention amplifies the fragment of IL-6 containing a plurality of important SNP sites, can flexibly select the detected sites and combinations according to the actual requirements, can play a role of one box for multiple purposes, and has the advantages of high primer specificity, low mismatching rate, primer length and T in the kit_mThe value design is reasonable, the amplification product is convenient to detect and separate, the amplification efficiency is greatly improved, the amplification cost is reduced, and the method has good popularization value.

Description

Kit for detection

Technical Field

The invention relates to a detection kit, belongs to the field of biology, and particularly relates to the field of biological detection.

Background

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in body defense, acute, immune, and hematopoietic responses. IL-6 is mainly produced by macrophages, T cells, B cells, and the like. It can regulate the growth and differentiation of various cells, regulate immune response, acute phase reaction and hemopoietic function, and play an important role in the anti-infection immune response of organism. IL-6 has been significantly altered in a variety of diseases. Dysregulation of IL-6 expression can cause a number of diseases, the clinical manifestations of which are mainly increased IL-6 levels at the onset. The elevated level of IL-6 is closely related to the active phase of the disease, the changes in tumor development, the extent of rejection and the effectiveness of the treatment, and therefore, the measurement of IL-6 levels in the patient's body fluids reflects the changes in the patient's condition.

The gene of IL-6 has two forms of DNA repetitive sequence polymorphism and Single Nucleotide Polymorphism (SNP), researches prove that polymorphism exists in a non-coding region and a 3 'end of a 5' end of the IL-6 gene, and the single nucleotide polymorphism of the IL-6 gene is related to various diseases such as hypertension, coronary heart disease, diabetes mellitus and the like. The existing IL-6 detection is the detection of single SNP site, and cannot simultaneously amplify and detect a plurality of SNP sites of IL-6. Therefore, the design and development of a detection kit has important clinical use value.

Disclosure of Invention

In view of the above problems of the prior art, the present invention has an object to provide a detection kit capable of detecting a plurality of SNP sites of an IL-6 gene.

In order to achieve the above object, the technical scheme of the detection kit adopted by the invention is as follows:

the kit for detection is used for amplifying the IL-6 gene and sequencing an amplification product, and comprises IL-6 upstream and downstream amplification primers, DNA polymerase, amplification buffer and dNTPs.

The kit for detection is mainly used for carrying out universal amplification on sections of IL-6 genes with various clinical biological meanings, and amplification products comprise a plurality of Single Nucleotide Polymorphism (SNP) sites of IL-6 with important influence. The G/C replacement at the 174 site, the C/G replacement at the 572 site and the A/G replacement at the 597 site are proved to be determinants of various diseases, and the replacements can influence the recognition of a plurality of restriction enzymes, so that the detection of the IL-6 gene containing the segment after amplification has accurate guiding significance for the disease onset type, risk prediction of the disease onset rate, diagnosis and treatment after the disease onset.

Preferably, one side sequence of the IL-6 to be amplified of the kit is shown in a sequence table SEQ ID NO: 1 is shown.

Preferably, the other side sequence of the IL-6 to be amplified in the kit is shown in a sequence table SEQ ID NO: 2, respectively.

Preferably, the method is used for amplifying a polypeptide shown as SEQ ID NO: the sequence of the upstream primer of the fragment shown in 1 is shown as a sequence table SEQ ID NO. 3.

Preferably, the method is used for amplifying a polypeptide shown as SEQ ID NO: 2 is shown in a sequence table SEQ ID NO. 4.

The amplification method of the invention is specific to the IL-6 gene, the specific primer can accurately identify and specifically amplify the corresponding target fragment under the coexistence state of a plurality of template DNAs, the amplification specificity is high, the non-specific bands are few, the amplified fragment can be simply separated and then is subjected to subsequent detection, and the applicability is strong.

Preferably, the detection sites of the detection kit include 174 sites, 572 sites and 597 sites.

In the method of the present invention, since simultaneous amplification of a plurality of fragments is involved, it is recommended to minimize the influence of interference factors on the amplification system, and therefore, it is preferable to amplify the extracted human whole blood DNA as an amplification template, and if the human whole blood DNA is directly used as a template without extraction, a common buffer needs to be replaced with a whole blood amplification buffer.

Preferably, the whole blood amplification buffer contains 100mmol/L Tris-HCl, 50mmol/L KCl and has pH of 9.3-9.5.

The amplification system in the amplification method is a 50 mu L PCR system, and specifically comprises the following steps:

1×PCR buffer 2μL；

Mg²⁺(25mmol/L)1μL；

dNTPs(25mmol/L)0.6μL；

taq enzyme (5U/. mu.L) 0.3. mu.L;

2 μ L of template DNA;

1 μ L of each of the upstream and downstream primers (10 μmol/L);

sterile double distilled water is filled to 50 mu L.

The system is a simple amplification system, does not contain any dye, probe and the like, and if the amplification product has subsequent experimental requirements, corresponding reagents can be added into the system according to the specific requirements.

The amplification condition of the method is specifically pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 90s, and extension at 72 ℃ for 50s for 30 cycles. The present conditions are preferred conditions of the present invention, and the step adjustment based on the amplification conditions of the present invention should be considered to fall within the scope of the present invention as long as the target amplification product can be obtained, and the amplification method of the present invention is not limited to the specific amplification conditions described above.

Another object of the present invention is to provide a method for detecting IL-6 gene in a universal manner, which comprises using the above-mentioned detection kit. The kit adopts the amplification method, is efficient and accurate, greatly reduces the use of amplification reagents, reduces the volume of an amplification system, meets the requirement of environmental protection and conservation while amplifying efficiently, reduces the amplification cost, and provides a new idea for the wide popularization of gene sequencing.

The amplified gene fragment of IL-6 in the invention can be used as a basis for further identification and diagnosis in various aspects such as gene screening, disease susceptibility, medication guidance and the like, and has important significance for the research and clinical development of the gene.

Compared with the prior art, the kit has the advantages that the IL-6 fragment containing a plurality of important SNP sites is amplified, the detected sites and combinations can be flexibly selected according to actual needs, the kit can play a role in one box for multiple purposes, the specificity of primers in the kit is high, the mismatching rate is low, the length and Tm value of the primers are reasonably designed, the amplification products are convenient to detect and separate, the amplification efficiency is greatly improved, the amplification cost is reduced, and the popularization value is good.

Detailed Description

The detection kit provided by the present invention will be described in detail and fully below with reference to examples. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

The detection kit designed in this example has a universal amplification primer designed for the structure of IL-6, and can simultaneously amplify a segment including gene polymorphism sites, wherein the segment includes 174 sites, 572 sites, 597 sites and the like. The amplified product can simultaneously detect the gene polymorphism of the sites.

The kit comprises IL-6 upstream and downstream amplification primers, DNA polymerase, amplification buffer solution and dNTPs, wherein the Taq DNA polymerase is Ex Taq DNA polymerase of TaKaRa company.

The DNA template used in the kit of this embodiment is a processed extracted DNA template, and it is necessary to previously process the collected human whole blood or tissue. If the sample to be amplified is an unprocessed whole blood sample, a buffer solution for PCR of the whole blood sample is adopted, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has the pH value of 9.3-9.5.

The gene sequence of the IL-6 gene to be amplified in this example (SEQ ID NO: 1) is as follows:

AGAAGCCACGCGGTGGCAAAAAGGAGTCACACACTCCACCTGGAGACGCCTTGAAGTAACTGCACGAAATTTGAGGATGGCCAGGCAGTTCTACAACAGCCGCTCACAGGGAGAGCCAGAACACAGAAGAACTCAGATGACTGGTAGTATTACCTTCTTCATAATCCCAGGCTTGGGGGGCTGCGATGGAGTCAGAGGAAACTCAGTTCAGAACATCTTTGGTTTTTACAAATACAAATTAACTGGAACGCTAAATTCTAGCCTGTTAATCTGGTCACTGAAAAAAAATTTTTTTTTTTTCAAAAAACATAGCTTTAGCTTATTTTTTTTCTCTTTGTAAAACTTCGTGCATGACTTCAGCTTTACTCTTTGTCAAGACATGCCAAAGTGCTGAGTCACTAATAAAAGAAAAAAAGAAAGTAAAGGAAGAGTGGTTCTGCTTCTTAGCGCTAGCCTCAATGACGACCTAAGCTGCACTTTTCCCCCTAGTTGTGTCTTGC

the downstream gene sequence (SEQ ID NO: 2) of the IL-6 gene to be amplified in this example is as follows:

TGGCCAGGCAGTTCTACAACAGCCGCTCACAGGGAGAGCCAGAACACAGAAGAACTCAGATGACTGGTAGTATTACCTTCTTCATAATCCCAGGCTTGGGGGGCTGCGATGGAGTCAGAGGAAACTCAGTTCAGAACATCTTTGGTTTTTACAAATACAAATTAACTGGAACGCTAAATTCTAGCCTGTTAATCTGGTCACTGAAAAAAAATTTTTTTTTTTTCAAAAAACATAGCTTTAGCTTATTTTTTTTCTCTTTGTAAAACTTCGTGCATGACTTCAGCTTTACTCTTTGTCAAGACATGCCAAAGTGCTGAGTCACTAATAAAAGAAAAAAAGAAAGTAAAGGAAGAGTGGTTCTGCTTCTTAGCGCTAGCCTCAATGACGACCTAAGCTGCACTTTTCCCCCTAGTTGTGTCTTGCCATGCTAAAGGACGTCACATTGCACAATCTTAATAAGGTTTCCAATCAGCCCCACCCGCTCTGGCCCCACCCTCACC

two pairs of amplification primers are designed according to the sequences, and the primer sequences are specifically as follows:

third, PCR amplification

Selecting a 50. mu.L PCR system in which

1×PCR buffer 2μL；

Mg²⁺(25mmol/L)1μL；

dNTPs(25mmol/L)0.6μL；

Taq enzyme (5U/. mu.L) 0.3. mu.L;

2. mu.L of each template DNA;

1 μ L of each of the upstream and downstream primers (10 μmol/L);

sterile double distilled water is filled to 50 mu L.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 90s, and extension at 72 ℃ for 50s, for 40 cycles.

Fourth, product verification

2% agarose gel electrophoresis is selected, and the electrophoresis result proves that the amplification result is correct. And recovering the electrophoresis result gel and then sequencing, wherein the sequencing result is consistent with the sequence of the target product.

And (3) detecting the amplification product at 174 sites, 572 sites and 597 sites, wherein the genotype detection result of each site accords with the distribution rule. Further studies are needed regarding the relevance of each site to the disease.

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A kit for detection is characterized by being used for amplifying an IL-6 gene and sequencing an amplification product, and comprising IL-6 upstream and downstream amplification primers, DNA polymerase, an amplification buffer solution and dNTPs.

2. The detection kit according to claim 1, wherein the IL-6 side sequence to be amplified in the kit is represented by SEQ ID NO: 1 is shown.

3. The detection kit according to claim 1, wherein the other side sequence of IL-6 to be amplified in the kit is represented by SEQ ID NO: 2, respectively.

4. The detection kit according to claim 1, wherein the kit is used for amplifying a nucleic acid sequence shown in SEQ ID NO: the sequence of the upstream primer of the fragment shown in 1 is shown as a sequence table SEQ ID NO. 3.

5. The detection kit according to claim 1, wherein the kit is used for amplifying a nucleic acid sequence shown in SEQ ID NO: 2 is shown in a sequence table SEQ ID NO. 4.

6. The detection kit according to claim 1, wherein the detection sites of the detection kit include 174, 572 and 597 sites.

7. The detection kit according to claim 1, wherein the amplification system used in the amplification method is a 50 μ L PCR system, and specifically comprises:

1×PCR buffer 2μL；

Mg²⁺(25mmol/L)1μL；

dNTPs(25mmol/L)0.6μL；

taq enzyme (5U/. mu.L) 0.3. mu.L;

2 mu L of template DNA;

1 μ L of each of the upstream and downstream primers (10 μmol/L);

sterile double distilled water is filled to 50 mu L.

8. The detection kit according to claim 1, wherein the specific amplification conditions of the amplification method are: pre-denaturation at 94 ℃ for 5 min; then denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 90s, and extension at 72 ℃ for 50s, for 40 cycles.

9. The detection kit according to claim 1, wherein the amplification method further comprises a whole blood amplification buffer solution, the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl, and has a pH of 9.3 to 9.5.

10. A method for detecting IL-6 gene in general, which comprises detecting the IL-6 gene using the detection kit according to any one of claims 1 to 9.