CN113684279A

CN113684279A - Primer group, kit and detection method for diagnosing osteosarcoma

Info

Publication number: CN113684279A
Application number: CN202111181385.2A
Authority: CN
Inventors: 张海方; 谢宗刚; 陈志豪; 杜鸿; 韩明霄; 李柳炳; 李自愿
Original assignee: Nuclear Industry General Hospital
Current assignee: Nuclear Industry General Hospital
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2021-11-23

Abstract

The invention discloses a primer group, a kit and a detection method for diagnosing osteosarcoma, belonging to the technical field of nucleic acid detection, wherein the primer group comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; the kit comprises a primer group for rapidly detecting osteosarcoma specific micro RNA. The invention applies the principle of the Poly (A) tailing method, designs specific primers and applies the real-time fluorescence quantitative PCR technology to establish the method for quickly detecting the osteosarcoma serum specific micro RNA, the method only needs one-time sample adding, one reaction tube and one reaction system to complete the detection of the osteosarcoma serum specific micro RNA of multiple samples within 2 hours, and the method has the advantages of quickness, high efficiency, accuracy and the like, saves the sample adding time and the experiment cost, has the advantages of quickly and effectively detecting multiple samples at one time, and can provide effective data for clinical detection in time.

Description

Primer group, kit and detection method for diagnosing osteosarcoma

Technical Field

The invention relates to the technical field of nucleic acid detection, in particular to a primer group, a kit and a detection method for diagnosing osteosarcoma.

Background

Osteosarcoma is a rapidly developing primary malignant tumor of the bone. The combined application of surgery, chemotherapy and radiotherapy obviously improves the survival rate of osteosarcoma patients, but the prognosis of bone tumor patients is still poor. Most osteosarcoma patients eventually die from lung metastases. The research results prove that the occurrence and the evolution of osteosarcoma are closely related to gene mutation and expression level. Therefore, the research on the potential gene molecule expression mechanism of osteosarcoma, the search for potential biomarkers and therapeutic targets are crucial to the early diagnosis of diseases and the proposal of new therapeutic schemes. The microRNA has a primary application in early diagnosis of tumor as a serum molecular target, but no kit for serum detection of osteosarcoma related microRNA exists in the market.

The method is important for guiding clinical diagnosis and treatment scheme formulation for early diagnosis of osteosarcoma, and can effectively reduce tumor spread and prolong the survival time of patients. Tissue biopsy is the traditional means of diagnosing osteosarcoma. At present, the tissue biopsy procedure generally adopted in domestic clinic takes about 14-21 days as a whole, the method takes long time, and the method can not meet the clinical requirements for rapid diagnosis and treatment plan of osteosarcoma. At the same time, the risk of tumor spread due to tissue sampling is more difficult to control. Therefore, the traditional diagnosis means can not realize the rapid diagnosis of osteosarcoma within hours and avoid the risk of cancer focus diffusion caused by invasive operation.

In recent years, as the research on microRNAs is gradually deepened, the potential of microRNAs as cancer diagnosis and intervention targets is gradually revealed. Based on the diagnosis of serum micro RNA, the specific recognition nucleic acid sequence can be used for diagnosing cancer by the PCR technology, and the method has the advantages of simplicity, rapidness, specificity and sensitivity. And the method establishes a series of rapid detection and diagnosis platforms in the aspects of clinical tumor diagnosis and treatment, and comprises the following steps: identification of cancer related miRNA such as liver cancer and breast cancer. However, no kit for detecting serum microRNA by using PCR technology exists so far, and particularly no kit for detecting osteosarcoma serum specific microRNA exists.

Disclosure of Invention

The invention aims to provide a primer group, a kit and a detection method for diagnosing osteosarcoma, which are used for solving the problems in the prior art, and can be used for quickly, efficiently and accurately detecting serum specific micro RNA of osteosarcoma, so that the detection time is greatly shortened, the detection cost is saved, and the detection efficiency is improved.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a primer group for rapidly detecting osteosarcoma specific micro RNA, which comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; the specific micro RNA is hsa-miR-338-3 p.

The invention also provides application of the primer group in preparation of products for detecting osteosarcoma specific micro RNA.

Further, the product can be used for diagnosing osteosarcoma by measuring the expression level of hsa-miR-338-3p in a sample.

Further, the sample comprises serum.

Further, the product comprises a product for detecting the level of hsa-miR-338-3p by fluorescent quantitative PCR to diagnose osteosarcoma.

Further, the product specifically comprises the following use methods:

step 1: extracting RNA in a sample to be detected;

step 2: synthesizing cDNA by using the extracted RNA as a template, and performing fluorescent quantitative PCR by using the primer group;

and step 3: and (3) carrying out melting curve analysis on the amplification product, and identifying the osteosarcoma serum specific micro RNA molecule hsa-miR-338-3p in the sample by taking a negative quality control product as a control.

Further, the reaction system of the fluorescent quantitative PCR in the step 2 is composed ofComprises the following components: 2U/. mu.L Ace Taq enzyme 1. mu.L, 2.5mM dNTP mix 4. mu.L, 1.5mM Mg²⁺3 mu L, 2.5U/. mu.L RNaseH 1.5. mu.L, SYBRGreen I0.5. mu.L, each primer in the primer set is 0.2. mu.L, sample DNA 3. mu.L and ddH₂O is supplemented to 20 mu L; the concentration of the sample DNA is 20-100 ng/mL.

Further, the reaction procedure of the fluorescent quantitative PCR in the step 2 is as follows: at 95 ℃ for 10min, at 95 ℃ for 10s, at 60 ℃ for 30s, for 40 cycles.

Further, the melting curve analysis program in step 3 is: denaturation at 95 ℃ for 15 s; the temperature rise range is 60-95 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out.

The invention also provides a kit for rapidly detecting the osteosarcoma specific micro RNA, and the kit comprises the primer group for rapidly detecting the osteosarcoma specific micro RNA.

The invention discloses the following technical effects:

(1) the invention establishes a method for rapidly detecting the osteosarcoma serum specific micro RNA by applying the principle of a Poly (A) tailing method, designing a specific primer and applying a real-time fluorescence quantitative PCR technology, and the method only needs one-time sample adding, one reaction tube and one reaction system to complete the detection of the osteosarcoma serum specific micro RNA of multiple samples within 2 hours, thereby having the advantages of rapidness, high efficiency, accuracy and the like, greatly shortening the detection time, saving the detection cost and improving the detection efficiency.

(2) Compared with other PCR methods, such as probe multiplex PCR, the primer and the probe need to be designed and synthesized simultaneously, and the modifying group of the taqman probe is expensive and is easy to interfere with each other. The invention adopts a real-time fluorescent quantitative PCR melting curve method for designing specific primers, which is an economic, rapid and effective method, and the whole reaction process is totally closed and stable.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described 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 without creative efforts.

FIG. 1 is a graph of the real-time fluorescent quantitative PCR amplification in example 2, wherein the gray line is the internal reference U6 and the black line is hsa-miR-338-3 p;

FIG. 2 is a melting curve diagram of real-time fluorescent quantitative PCR in example 2, wherein the gray line is internal reference U6 and the black line is hsa-miR-338-3 p;

FIG. 3 is a comparison of the expression levels of hsa-miR-338-3p in the serum of healthy human and osteosarcoma patients in example 2.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

Composition of kit for rapidly detecting osteosarcoma specific micro RNA and detection method

1. Composition of the kit

(1) A primer group: the amplification primer pair for detecting osteosarcoma specific micro RNA, hsa-miR-338-3p, comprises a specific primer hsa-miR-338-3p-F and a universal downstream primer which are designed according to the principle of a Poly (A) tailing method, namely, an ' A ' is added at the 3' end of a PCR product, so that the specific primer can be connected with a primer with an outstanding ' T '.

The amplification primer pair of the hsa-miR-338-3p gene is as follows:

hsa-miR-338-3p-F(SEQ ID NO:1)：5’-CGCAACAATATCCTGGTGCTGAGTG-3’；

universal downstream primer (SEQ ID NO: 2): 5'-AACGCTTCACGAATTTGCGT-3' are provided.

The PCR primers were synthesized by Beijing Edley Biotechnology Ltd.

(2) Process for preparing reagent kit

Prepare 20. mu.L of PCR reaction system (as shown in Table 1):

TABLE 120 μ L PCR reaction System

2. Method for detecting osteosarcoma serum specific micro RNA by kit

Step 1: extracting nucleic acid of a sample: extracting micro RNA in a serum sample to be detected by adopting a micro RNA extraction kit (purchased from Edele Biotechnology Ltd.);

step 2: synthesizing cDNA by using the extracted micro RNA as a template and a miRNA first strand synthesis kit (purchased from Edley Biotechnology Co., Ltd.); the concentration of the cDNA template is 20-100 ng/mL; taking U6 as a relative quantitative internal reference, performing real-time fluorescence quantitative PCR by using the primer group, adding a real-time fluorescence quantitative PCR reaction system into a 0.2mL optical PCR reaction tube as shown in the table 1, and slightly covering the cover;

the PCR reaction program is: at 95 ℃ for 10min, at 95 ℃ for 10s, at 60 ℃ for 30s, for 40 cycles.

And step 3: and (3) carrying out melting curve analysis on the amplified product, taking a negative quality control based on the expression level of a normal person as a control, and taking U6 as a relative quantitative internal reference to identify osteosarcoma specific micro RNA, hsa-miR-338-3p, in the serum sample. The melting curve analysis program was: denaturation at 95 ℃ for 15 s; the temperature rise range is 60-95 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out.

Example 2

Method for detecting osteosarcoma serum specific micro RNA by using kit described in example 1

1. Sample(s)

Clinical samples of normal human serum were collected, and cDNA was obtained by the method described in example 1 to prepare negative cDNA samples. The clinical sample used in this example is serum sample of osteosarcoma patients, and there is no limitation on osteosarcoma tissue typing and lesion site.

2. Detection of osteosarcoma specific microRNA, hsa-miR-338-3p in serum

Firstly, preparing PCR reaction solution according to table 1 in example 1, arranging 3 parallel reaction solutions, subpackaging the reaction solutions in 2mL reaction tubes, respectively adding a template to be detected, covering a tube cover, putting the tube cover into an Applied Biosystems StepOnePlus 96 fluorescent real-time PCR instrument, and carrying out PCR reaction by adopting the following reaction procedures: at 95 ℃ for 10min, at 95 ℃ for 10s, at 60 ℃ for 30s, for 40 cycles. The real-time fluorescent PCR amplification curve is shown in FIG. 1.

After the amplification is finished, an Applied biosystems StepOneplus real Time PCR Instrument is used for detecting a melting curve, and the melting process is 95 ℃ denaturation for 15 s; the temperature rise range is 60-95 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out. The results of melting curve analysis are shown in FIG. 2.

3. Statistical method

Each experiment was repeated 3 times, and the data were expressed as mean ± standard deviation, and were statistically analyzed using SPSS21.0 statistical software. The difference between the two was considered statistically significant when P <0.05 using the t-test.

4. Results of the experiment

As shown in FIG. 3, the expression level of the osteosarcoma-specific microRNA, hsa-miR-338-3P, in the blood of osteosarcoma patients is decreased compared with that of the normal population, and the difference is statistically significant (P < 0.05).

Through the above experimental results, it is found that: in the same reaction system, the detection of the osteosarcoma serum specific microRNA is completed within 2 hours, and the specific primer designed by the principle of Poly (A) tailing method is used for detecting the osteosarcoma serum specific microRNA, hsa-miR-338-3p, through real-time fluorescence PCR, so that osteosarcoma patients and normal people can be accurately distinguished.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

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Claims

1. A primer group for rapidly detecting osteosarcoma specific micro RNA is characterized by comprising an upstream primer with a nucleotide sequence shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; the specific micro RNA is hsa-miR-338-3 p.

2. The application of the primer group in claim 1 in preparing products for detecting osteosarcoma specific micro RNA.

3. The use of claim 2, wherein the product is used to diagnose osteosarcoma by measuring the expression level of hsa-miR-338-3p in a sample.

4. The use of claim 3, wherein the sample comprises serum.

5. The use according to claim 3, wherein the product comprises a product for diagnosing osteosarcoma by detecting the level of hsa-miR-338-3p by fluorescent quantitative PCR.

6. The use according to claim 5, wherein the product comprises in particular the following method of use:

step 1: extracting RNA in a sample to be detected;

7. The use of claim 6, wherein the reaction system of the fluorescence quantitative PCR in the step 2 is composed of the following components: 2U/. mu.L Ace Taq enzyme 1. mu.L, 2.5mM dNTP mix 4. mu.L, 1.5mM Mg²⁺3 mu L, 2.5U/. mu.L RNaseH 1.5. mu.L, SYBRGreen I0.5. mu.L, each primer in the primer set is 0.2. mu.L, sample DNA 3. mu.L and ddH₂O is supplemented to 20 mu L; the concentration of the sample DNA is 20-100 ng/mL.

8. The use of claim 6, wherein the reaction procedure of the fluorescent quantitative PCR in step 2 is as follows: at 95 ℃ for 10min, at 95 ℃ for 10s, at 60 ℃ for 30s, for 40 cycles.

9. The use of claim 6, wherein the melting curve analysis procedure in step 3 is: denaturation at 95 ℃ for 15 s; the temperature rise range is 60-95 ℃, the temperature rise rate is 0.11 ℃/s, fluorescence signals are continuously collected for 5 times/DEG C, and melting curve analysis is carried out.

10. A kit for rapidly detecting osteosarcoma specific microRNA, which comprises the primer set for rapidly detecting osteosarcoma specific microRNA according to claim 1.