阅读说明：本技术 血清中环状circZKSCAN1基因的非诊断性荧光定量检测方法 (Non-diagnostic fluorescent quantitative detection method for circular circZKSCAN1 gene in serum ) 是由 梁浩凡 缪辉来 程海兵 于 2020-12-25 设计创作，主要内容包括：本发明提供了一种血清中环状circZKSCAN1基因的非诊断性荧光定量检测方法,通过荧光定量反应PCR检测正常肝脏血清和肝癌患者血清,结果显示,肝癌患者血清Exosome中的环状circZKSCAN1基因表达量明显较高。本发明提供的检测方法能够有效检测肝癌患者血清中Exosome的表达,对于肝癌的检出率达到95％以上,达到肝癌早期诊断的目的,填补目前在肝癌早期诊断领域的市场空白,为肝癌的早期临床诊断提供一种理想的基因检测方法。(The invention provides a non-diagnostic fluorescent quantitative detection method of a circular circZKSCAN1 gene in serum, which detects normal liver serum and liver cancer patient serum through fluorescent quantitative reaction PCR, and the result shows that the circular circZKSCAN1 gene expression in liver cancer patient serum Exosome is obviously higher. The detection method provided by the invention can effectively detect the expression of Exosome in serum of a liver cancer patient, the detection rate of liver cancer reaches more than 95%, the aim of early diagnosis of liver cancer is fulfilled, the market blank in the field of early diagnosis of liver cancer at present is filled, and an ideal gene detection method is provided for early clinical diagnosis of liver cancer.)

1. The non-diagnostic fluorescent quantitative detection method of the circular circZKSCAN1 gene in serum is characterized by comprising the following steps:

1) extracting Exosome: extracting exosomes in serum, specifically operating as follows:

(a) placing 1mL of serum in a 1.5mL centrifuge tube, centrifuging for 20min at 2000g, removing larger cell debris, and taking supernatant;

(b) adding 250 mu L of 5X PEG-8000NaCl solution into every 1mL of the supernatant obtained in the step (a); mixing once every 20-30 min, carrying out 3-5 times, and standing at 4 ℃ for 8-24 h to ensure that the Exosome is fully settled;

(c) after sedimentation, centrifuging at 4 ℃ for 30min at 10000g, and removing supernatant;

(d) centrifuging at 4 deg.C for 5min at 10000g, and discarding the residual liquid;

(e) obtaining precipitated Exosome;

2) and (3) RNA extraction: extracting RNA in the exosomes obtained in the step 1);

3) removing residual genomic DNA from the extracted RNA: adding reaction liquid into the RNA extracted in the step 2), and removing residual genome DNA after digestion and inactivation;

4) reverse transcription of RNA into cDNA: adding the RNA in the step 3) into a PCR tube as a template, and performing reverse transcription to form cDNA in a PCR system by adopting a random reverse transcription primer;

5) adopting a fluorescent quantitative reaction Real-time PCR kit to prepare a reaction system, and detecting the circular circZKSCAN1 gene in the serum Exosome of a normal liver serum and liver cancer patient; the specific detection reaction system is as follows:

wherein, the upstream and downstream primers are respectively:

upstream primer of 5'TACCGCCCCGATAGTGGAGA 3'

A downstream primer: 5'TGAAGTGGGACTGGGTGGC 3';

the fluorescent quantitative PCR reaction conditions are as follows: denaturation at 95 ℃ for 5 min; 10 seconds at 95 ℃ and 35 seconds at 60 ℃; 40 cycles, and collecting the fluorescence signal at a temperature of 60 ℃ to 95 ℃ and then analyzing.

2. The non-diagnostic fluorescent quantitative determination method according to claim 1, characterized in that the preparation method of 5X PEG-8000NaCl solution: weighing 8.766g of NaCl and 800050g and dissolving in 200mL of Milli-Q pure water; extinguishing by damp heat at 121 deg.C for 30 min; 5X PEG-8000NaCl solution was obtained and stored at 4 ℃.

3. The non-diagnostic quantitative fluorescence detection method according to claim 1, wherein step 2): extracting RNA in the exosomes obtained in the step 1), and specifically operating as follows:

(a) adding 1mL of Trizol into 1mL of Exosome extracted from 1mL of serum;

(b) adding 200 μ L chloroform, shaking vigorously for 15 s, and standing at room temperature for 15 min;

(c) centrifuging at 12000g for 15min at 4 deg.C, separating the solution into three layers, dissolving RNA in the water phase, and transferring the water phase to another new RNase free EP tube;

(d) adding isopropanol with the volume of 1 time, and fully and uniformly mixing by vortex;

(e) centrifuging at 12000g for 10min at 4 deg.C, collecting RNA precipitate at the bottom of the tube, and discarding the supernatant;

(f) adding 1mL of 75% ethanol, slightly inverting by hand, centrifuging at 12000g for 5min, and discarding the supernatant;

(g) air dried at room temperature, and 20. mu.L of DEPC water was added to dissolve the precipitate.

4. The non-diagnostic quantitative fluorescence detection method according to claim 3, wherein step 3): adding reaction solution into the RNA extracted in the step 2), digesting the RNA at 37 ℃ for 30min, adding 0.5 mu L of EDTA, and inactivating the RNA at 65 ℃ for 10min to remove residual genome DNA.

5. The non-diagnostic quantitative fluorescence detection method according to claim 4, wherein step 4): adding the RNA in the step 3) into a PCR tube as a template, and performing reverse transcription to form cDNA in a PCR system by adopting a random reverse transcription primer;

the PCR system of reverse transcription is:

RNase free ddH₂complement O up to 1 to 10 μ L;

the reaction conditions are as follows: 5min at 25 ℃, 20min at 42 ℃, 5min at 85 ℃ and 2min at 4 ℃.

Technical Field

The invention relates to the technical field of early diagnosis of liver cancer, in particular to a non-diagnostic fluorescent quantitative detection method of a cyclic circZKSCAN1 gene in serum.

Background

Circular RNA (circRNA) in organisms is a type of RNA with a specific unknown function and is objectively abundant. The circular RNA is formed by cutting precursor RNA and then connecting linear RNA in a head-to-tail mode, the existing part of objective RNA is ignored in the previous research due to the limitation of the technical level, and researchers really find that a large amount of circular RNA molecules exist in organisms along with the development of deep RNA sequencing and scale bioinformatics technology, and the circular RNA is very stable in the organisms due to the formation of closed circles. Specific functions of circularized RNA are not clear, and at present, only a few hypotheses exist that 1) circular RNA can be used as "sponge absorbing miRNA to inhibit the functions of the miRNA; 2) the circRNA directly regulates and controls other RNA level through base complementary pairing; 3) the circRNA can be combined with protein, inhibit protein activity, recruit components of protein complex or regulate protein activity; 4) circRNA can also be used as a template for translation to direct protein synthesis.

While Exosomes originate from endocytosed small membrane vesicles and can be secreted by many types of cells, it and circRNA are hot research spots in recent years. It was found that exosomes in the serum of liver cancer patients contain large amounts of circRNA and are extremely stable. In areas or countries with high incidence of liver cancer, if early diagnosis and early treatment can be achieved, the method will bring far-reaching significance to prevention and treatment of tumors.

In view of the above, it is necessary to provide a technical solution to the above problems.

Disclosure of Invention

The invention aims to: provides a non-diagnostic fluorescent quantitative detection method of the circular circZKSCAN1 gene in serum, and fills the market gap in the field of early diagnosis of liver cancer at present. The detection method of the kit provided by the invention can effectively monitor the expression of exosomes in the serum of a patient, and achieves the aim of early diagnosis of liver cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the non-diagnostic fluorescent quantitative detection method of the circular circZKSCAN1 gene in serum comprises the following steps:

1) extracting Exosome: extracting exosomes in serum, specifically operating as follows:

(a) placing 1mL of serum in a 1.5mL centrifuge tube, centrifuging for 20min at 2000g, removing larger cell debris, and taking supernatant;

(b) adding 250 mu L of 5X PEG-8000NaCl solution into every 1mL of the supernatant obtained in the step (a); mixing once every 20-30 min, carrying out 3-5 times, and standing at 4 ℃ for 8-24 h to ensure that the Exosome is fully settled;

(c) after sedimentation, centrifuging at 4 ℃ for 30min at 10000g, and removing supernatant;

(d) centrifuging at 4 deg.C for 5min at 10000g, and discarding the residual liquid;

(e) obtaining precipitated Exosome;

2) and (3) RNA extraction: extracting RNA in the exosomes obtained in the step 1);

3) removing residual genomic DNA from the extracted RNA: adding reaction liquid into the RNA extracted in the step 2), and removing residual genome DNA after digestion and inactivation;

4) reverse transcription of RNA into cDNA: adding the RNA in the step 3) into a PCR tube as a template, and performing reverse transcription to form cDNA in a PCR system by adopting a random reverse transcription primer;

5) adopting a fluorescent quantitative reaction Real-time PCR kit to prepare a reaction system, and detecting the circular circZKSCAN1 gene in the serum Exosome of a normal liver serum and liver cancer patient; the specific detection reaction system is as follows:

wherein, the upstream and downstream primers are respectively:

upstream primer of 5'TACCGCCCCGATAGTGGAGA 3'

A downstream primer: 5'TGAAGTGGGACTGGGTGGC 3';

the fluorescent quantitative PCR reaction conditions are as follows: denaturation at 95 ℃ for 5 min; 10 seconds at 95 ℃ and 35 seconds at 60 ℃; 40 cycles, and collecting the fluorescence signal at a temperature of 60 ℃ to 95 ℃ and then analyzing.

Preferably, the preparation method of the 5X PEG-8000NaCl solution comprises the following steps: weighing 8.766g of NaCl and 800050g and dissolving in 200mL of Milli-Q pure water; extinguishing by damp heat at 121 deg.C for 30 min; 5X PEG-8000NaCl solution was obtained and stored at 4 ℃.

Preferably, step 2): extracting RNA in the exosomes obtained in the step 1), and specifically operating as follows:

(a) adding 1mL of Trizol into 1mL of Exosome extracted from 1mL of serum;

(b) adding 200 μ L chloroform, shaking vigorously for 15 s, and standing at room temperature for 15 min;

(c) centrifuging at 12000g for 15min at 4 deg.C, separating the solution into three layers, dissolving RNA in the water phase, and transferring the water phase to another new RNase free EP tube;

(d) adding isopropanol with the volume of 1 time, and fully and uniformly mixing by vortex;

(e) centrifuging at 12000g for 10min at 4 deg.C, collecting RNA precipitate at the bottom of the tube, and discarding the supernatant;

(f) adding 1mL of 75% ethanol, slightly inverting by hand, centrifuging at 12000g for 5min, and discarding the supernatant;

(g) air dried at room temperature, and 20. mu.L of DEPC water was added to dissolve the precipitate.

Preferably, step 3): adding reaction solution into the RNA extracted in the step 2), digesting the RNA at 37 ℃ for 30min, adding 0.5 mu L of EDTA, and inactivating the RNA at 65 ℃ for 10min to remove residual genome DNA.

Preferably, step 4): adding the RNA in the step 3) into a PCR tube as a template, and performing reverse transcription to form cDNA in a PCR system by adopting a random reverse transcription primer;

the PCR system of reverse transcription is:

RNase free ddH₂complement O up to 1 to 10 μ L;

the reaction conditions are as follows: 5min at 25 ℃, 20min at 42 ℃, 5min at 85 ℃ and 2min at 4 ℃.

Compared with the prior art, the invention has the beneficial effects that: the invention detects the serum of normal liver and the serum of liver cancer patients by fluorescence quantitative reaction PCR, and the result shows that the expression level of the circular circZKSCAN1 gene in the serum Exosome of the liver cancer patients is obviously higher.

Drawings

FIG. 1 is a graph showing the results of the fluorescent quantitative detection of circular RNA of circZKSCAN1 in serum exosomes of example 1 of the present invention.

Detailed Description

In order to make the technical solutions and advantages of the present invention clearer, the present invention and its advantages will be described in further detail below with reference to the following detailed description and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

In the present invention, the kit for extracting RNA is produced by Life technologiesReagent kit; DNaseI reagent used for removing residual genome DNA in the extracted RNA is produced by the whole formula of gold company; the reverse transcription kit is a kit produced by Vazyme company; the fluorescent quantitative reaction kit is a Real-time PCR kit produced by Vazyme company.

Example 1

A non-diagnostic fluorescent quantitative detection method of a circular circZKSCAN1 gene in serum comprises the following steps:

1) extracting Exosome: extracting exosomes in serum, specifically operating as follows:

(a) placing 1mL of serum in a 1.5mL centrifuge tube, centrifuging for 20min at 2000g, removing larger cell debris, and taking supernatant;

(b) adding 250 mu L of 5X PEG-8000NaCl solution into every 1mL of the supernatant obtained in the step (a); mixing once every 20-30 min, carrying out 3-5 times, and standing at 4 ℃ for 8-24 h to ensure that the Exosome is fully settled;

(c) after sedimentation, centrifuging at 4 ℃ for 30min at 10000g, and removing supernatant;

(d) centrifuging at 4 deg.C for 5min at 10000g, and discarding the residual liquid;

(e) obtaining precipitated Exosome;

the preparation method of the 5X PEG-8000NaCl solution comprises the following steps: weighing 8.766g of NaCl and 800050g and dissolving in 200mL of Milli-Q pure water; extinguishing by damp heat at 121 deg.C for 30 min; obtaining 5X PEG-8000NaCl solution, preserving at 4 ℃;

2) and (3) RNA extraction: extracting RNA in the exosomes obtained in the step 1), and specifically operating as follows:

(a) adding 1mL of Trizol into 1mL of Exosome extracted from 1mL of serum;

(b) adding 200 μ L chloroform, shaking vigorously for 15 s, and standing at room temperature for 15 min;

(c) centrifuging at 12000g for 15min at 4 deg.C, separating the solution into three layers, dissolving RNA in the water phase, and transferring the water phase to another new RNase free EP tube;

(d) adding isopropanol with the volume of 1 time, and fully and uniformly mixing by vortex;

(e) centrifuging at 12000g for 10min at 4 deg.C, collecting RNA precipitate at the bottom of the tube, and discarding the supernatant;

(f) adding 1mL of 75% ethanol, slightly inverting by hand, centrifuging at 12000g for 5min, and discarding the supernatant;

(g) air drying at room temperature, adding 20 μ L DEPC water to dissolve the precipitate;

3) removing residual genomic DNA from the extracted RNA: adding reaction liquid into the RNA extracted in the step 2), digesting for 30min at 37 ℃, then adding 0.5 mu L EDTA, inactivating for 10min at 65 ℃ and removing residual genome DNA;

wherein the reaction solution is configured as follows: using DNaseI from RNase-free (all-purpose gold), the reaction mixture was prepared as follows according to the instructions:

4) reverse transcription of RNA into cDNA: adding the RNA in the step 3) into a PCR tube as a template, and performing reverse transcription to form cDNA in a PCR system by adopting a random reverse transcription primer;

the PCR system of reverse transcription is:

RNase free ddH₂complement O up to 1 to 10 μ L;

the reaction conditions are as follows: 5min at 25 ℃, 20min at 42 ℃, 5min at 85 ℃ and 2min at 4 ℃;

5) adopting a fluorescent quantitative reaction Real-time PCR kit to prepare a reaction system, and detecting the circular circZKSCAN1 gene in the serum Exosome of a normal liver serum and liver cancer patient; the specific detection reaction system is as follows:

wherein, the upstream and downstream primers are respectively:

upstream primer of 5'TACCGCCCCGATAGTGGAGA 3'

A downstream primer: 5'TGAAGTGGGACTGGGTGGC 3';

the fluorescent quantitative PCR reaction conditions are as follows: denaturation at 95 ℃ for 5 min; 10 seconds at 95 ℃ and 35 seconds at 60 ℃; 40 cycles, and collecting fluorescence signals at a temperature of 60 ℃ to 95 ℃ and then performing melting curve analysis.

The results of the detection of the circular circZKSCAN1 gene in normal liver serum and liver cancer patient serum exosomes are shown in figure 1. FIG. 1 shows that the expression level of cyclic circZKSCAN1 was significantly higher in serum exosomes (group T) of liver cancer patients relative to normal healthy people (group N). The detection result of the invention is the contrast result of 200 clinical liver cancer cases and normal healthy people, the detection rate of liver cancer reaches more than 95 percent, the aim of early diagnosis of liver cancer is achieved, the market blank in the field of early diagnosis of liver cancer at present is filled, and an ideal gene detection method is provided for the early clinical diagnosis of liver cancer.

Variations and modifications to the above-described embodiments may also occur to those skilled in the art, which fall within the scope of the invention as disclosed and taught herein. Therefore, the present invention is not limited to the above-mentioned embodiments, and any obvious improvement, replacement or modification made by those skilled in the art based on the present invention is within the protection scope of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

SEQUENCE LISTING

<110> Guangzhou Giselsa Biotech available Inc

<120> non-diagnostic fluorescent quantitative detection method of circular circZKSCAN1 gene in serum

<130> 2020

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> artificaial

<400> 1

TACCGCCCCGATAGTGGAGA 20

<210> 2

<211> 19

<212> DNA

<213> artificaial

<400> 2

TGAAGTGGGACTGGGTGGC 19