CN104818329B - OR4F3 and applications of the OR4F17 in HBV relevant disease diagnostic kits are prepared - Google Patents

Abstract

The present invention relates to technical field of biomedical detection, the present invention provides the new application of OR4F3 and OR4F17, application specifically in HBV relevant disease diagnostic kits are prepared, the kit and detection method of HBV relevant disease diagnosis are carried out invention further provides a kind of quantitative PCR detection using for OR4F3 and OR4F17.The kit and detection method of the present invention is easy, and reliably, the cycle is short, and specificity is high, is easy to clinical expansion.

Description

Application of OR4F3 and OR4F17 in the preparation of diagnostic kits for HBV-related diseases

technical field

The invention relates to the technical field of medical biological detection, in particular to the application of OR4F3 and OR4F17 in the preparation of a diagnostic kit for HBV-related diseases.

Background technique

Hepatitis B virus (HBV) is a hepatotropic complex DNA virus. Existing studies have shown that HBV is only susceptible to humans and orangutans. HBV infection can lead to chronic or acute hepatitis (chronichepatitis B, CHB), and chronic hepatitis infection is the main cause of liver cirrhosis (liver cirrhosis, LC), liver cancer (hepatocellular carcinoma, HCC) and liver failure (liver failure, LF) . Among them, liver cancer is the third most common malignant tumor after esophageal cancer and gastric cancer, which seriously endangers people's health. From a global perspective, the incidence of liver cancer ranks sixth, and the mortality rate ranks third. There are about 94 million chronic hepatitis B patients in China, and nearly 300,000 people die of liver cancer every year.

HBV-related diseases refer to related diseases caused by HBV infection, including but not limited to hepatitis B, liver fibrosis, liver cirrhosis, and liver cancer. Among these liver diseases, especially the diagnosis of liver cancer is of great significance to its treatment effect and quality of life. Some studies have pointed out that early liver cancer detected by peripheral blood-related indicators has a better treatment effect, significantly improved survival rate, and has little impact on the quality of life of patients. However, for patients with advanced liver cancer, not only does the survival rate not improve after treatment, but it has a greater impact on the quality of life of the patients. Therefore, the success of liver cancer treatment is closely related to the success of early diagnosis.

The currently accepted diagnostic index for liver cancer is α-fetoprotein (AFP). Alpha-fetoprotein is more abundant in the liver tissue of infants and patients with liver cancer, but less in normal human liver tissue. It is a mature diagnostic index for liver cancer. However, the area under the receiver operating characteristic curve (area under curve, AUC) is only about 81%.

The human olfactory receptor gene family is the largest gene family in the human genome, and OR4F3 and OR4F17 are members of the olfactory receptor gene family. Olfactory receptor proteins belong to G protein-coupled receptors (GPCRs), have a structure of 7 transmembrane domains, and are responsible for recognizing and mediating odor-related signal transduction. Studies have shown that olfactory receptor-related genes mutate in prostate cancer, and are related to the occurrence and development of prostate cancer, and have a certain predictive effect on prostate cancer ([1] Bettina Malnic, Paul A. Godfrey, and Linda B. Buck. The human olfactory receptorgene family.PNAS.Vol.101, no.8,2584-2589; [2]Elena Linardopoulouet.al.Transcriptional activity of multiple copies of a subtelomericallylocated olfactory receptor gene that is polymorphic in number and location.Human Molecular Gene 2001, Vol.10, No.21.2373-2383; [3] Weng J, WangJ, Hu X, et al. PSGR2, a novel G-protein coupled receptor, is overexpressed inhuman prostate cancer. International journal of cancer Journal international du cancer 2006 ; 118(6):1471-80 doi:10.1002/ijc.21527).

At present, there is no literature report on the application of OR4F3 and OR4F17 in the diagnosis of HBV-related diseases, especially liver cancer.

Contents of the invention

The purpose of the present invention is to provide diagnostic markers for HBV-related diseases, and the purpose of the present invention is also to provide new uses of OR4F3 and OR4F17, that is, the application in the preparation of diagnostic kits for HBV-related diseases.

The first aspect of the present invention provides the use of OR4F3 and OR4F17 as diagnostic markers for HBV-related diseases.

The second aspect of the present invention provides the application of OR4F3 and OR4F17 in the preparation of diagnostic reagents or diagnostic kits for HBV-related diseases.

The information of OR4F3 and OR4F17 described in the present invention is as follows:

OR4F3: Chromosome 5, NC_000005.10(181367287..181368225);

OR4F17: Chromosome 19, NC_000019.10 (107152..111690).

The application of OR4F3 and OR4F17 described in the present invention in the preparation of diagnostic reagents or diagnostic kits for HBV-related diseases, the diagnostic reagents are a combination of reagents for detecting the content of OR4F3 and OR4F17 in biological samples.

The diagnostic kit includes reagents for detecting the content of OR4F3 and OR4F17 in biological samples.

The reagents for detecting the content of OR4F3 and OR4F17 in biological samples are selected from the group consisting of PCR primers specific for detecting OR4F3 and OR4F17.

The PCR primers with detection specificity for OR4F3 and OR4F17 are as follows:

The biological sample is selected from: peripheral blood obtained from a subject.

The HBV-related diseases and HBV-related diseases refer to related diseases caused by HBV infection, including but not limited to hepatitis B, liver fibrosis, liver cirrhosis, and liver cancer.

The third aspect of the present invention provides a diagnostic kit for HBV-related diseases, which includes reagents for detecting the content of OR4F3 and OR4F17 in biological samples.

The diagnostic kit for HBV-related diseases of the present invention is composed of a reverse transcription system, a primer system and an amplification system, and the primer system includes:

PCR primers specific for detection of OR4F3 and OR4F17:

The biological sample is selected from: peripheral blood obtained from a subject.

The HBV-related diseases and HBV-related diseases refer to related diseases caused by HBV infection, including but not limited to hepatitis B, liver fibrosis, liver cirrhosis, and liver cancer.

The invention provides a diagnostic kit for HBV-related diseases.

The diagnostic kit of the present invention is a diagnostic kit for HBV-related diseases based on genomic DNA quantitative PCR detection, and can accurately calculate the copy numbers of OR4F3 and OR4F17 in the blood genome by quantitative PCR technology.

The fourth aspect of the present invention provides a method for detecting HBV-related diseases using the above-mentioned diagnostic kit. The detection method is shown in Figure 1, and specifically includes the following steps:

1. Extraction of blood genome total DNA;

2. A reagent for quantitatively detecting the copy numbers of OR4F3 and OR4F17 using primers, wherein the primers are shown in SEQ ID NO: 1-4;

3. Calculate the copy numbers of OR4F3 and OR4F17; preferably, the standard curve generated by the instrument's built-in software can be used to convert the matching calculation software for the copy numbers of OR4F3 and OR4F17 in the blood genome.

The total DNA in the peripheral blood genome of the sample is extracted by using the kit and detection method of the present invention, and the copy number of OR4F3 is firstly detected by using the kit. Among them, when the copy number of OR4F3 in the blood genome is <15.66/ml, the sample has a 99.94% possibility of being normal; when the copy number of OR4F3 in the blood genome is >56.08/ml, the sample has a 93.21% possibility of being normal Hepatitis; when the copy number of OR4F3 in the blood genome is >56.08/ml<15.66/ml, the sample has a 49.43% probability of hepatitis and a 50.67% probability of liver cancer. For samples whose copy number of OR4F3 is between 15.66/ml and 56.08/ml, the copy number of OR4F17 is determined. When the copy number of OR4F17 is less than 14.28/ml, the sample has a 77.92% possibility of being liver cancer, and a 22.08% probability of being liver cancer. The possibility is hepatitis; when the copy number of OR4F17>14.28/ml, the sample has a probability of 98.18% to be hepatitis (see Figure 2).

Beneficial protection and effect of the present invention are as follows:

The applicant obtained a large number of peripheral blood samples of HBV-related patients from the surgical hospital of Dongfang Hepatobiliary Hospital, which provided a strong guarantee for the research of the present invention.

In terms of technology, the detection of OR4F3 and OR4F17 is essentially a quantitative PCR detection of the blood genome, which has the characteristics of simple operation, sensitive detection, good specificity, and high repeatability, and has been increasingly used in clinical testing. in technology.

The basic detection method we adopt is quantitative PCR, which has been proved to be a highly sensitive and accurate detection method in modern experimental diagnostics, and the test technology is very mature. And we use the standard curve quantification method in this technology, which can accurately quantify the characteristic nucleic acid molecules in various samples.

Moreover, the AUC values of the two indexes OR4F3 and OR4F17 involved in the present invention are 81.1458% and 90.7262% respectively, which are higher in clinical reference value and reliability than alpha-fetoprotein. Therefore, this detection kit can improve the detection rate and accuracy of familial liver cancer, and has great significance for the clinical treatment of familial liver cancer.

The invention provides a quantitative PCR-based detection kit for familial liver cancer, which can diagnose HBV-related diseases by detecting the copy numbers of the olfactory genes OR4F3 and OR4F17 in the patient's peripheral blood genomic DNA. Its detection method is particularly convenient, with short cycle time and high sensitivity, and is an effective supplement to the current detection reagents.

Description of drawings

Figure 1: Diagnosis of HBV-related diseases by detecting the copy numbers of the olfactory genes OR4F3 and OR4F17 in the blood genome; A: Obtain peripheral blood from the patient to be tested; B: Obtain blood cells by centrifugation; C: Obtain peripheral white blood cells; D : total DNA extracted; E: DNA amplification using specific primers; F: analysis of amplification results using standard curve; G: correlation amplification curve.

Fig. 2: The detection result with the kit of the present invention; A: OR4F3 is used as the threshold value of the diagnostic index; B: OR4F17 is used as the threshold value of the diagnostic index.

Figure 3: The copy numbers of OR4F3 and OR4F17 genes measured in peripheral blood samples of patients with hepatitis B (CHB), peripheral blood samples of patients with liver cirrhosis (LC) and peripheral blood samples of patients with hepatitis B liver cancer (HBV-HCC).

Figure 4: Photos of blood sample collection and processing; the circles represent lymphocytes.

Figure 5: Difference detection results of OR4F3 (A) and OR4F17 (B) gene copy numbers in blood genomes of normal people and HBV-related patients.

Detailed ways

Now, the present invention will be described in detail in conjunction with the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

The reagents and raw materials used in the present invention are commercially available or can be prepared according to literature methods. The experimental method that does not indicate specific conditions in the following examples, generally according to conventional conditions such as the conditions described in Sambrook et al. , or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Example 1:

In the early stage, we used the high-throughput comparative genome hybridization chip (comparative genome hybridization, CGH) to screen the genes with highly variable copy numbers in the peripheral blood genome of patients with hepatitis B liver cancer, namely OR4F3 and OR4F17.

Then we collected peripheral blood samples from 5 normal people as controls, peripheral blood samples from 10 patients with hepatitis B (CHB), peripheral blood samples from 10 patients with liver cirrhosis (LC) and peripheral blood samples from 10 patients with hepatitis B-hepatic cancer (HBV-HCC) Specimens, genomic DNA was extracted, and primers were designed for PCR verification. It was found that compared with the control, the copy numbers of the two genes OR4F3 and OR4F17 were significantly increased in varying degrees in the tested samples (see Figure 3).

The results suggest that OR4F3 and OR4F17 may be involved in the occurrence and development of HBV liver cancer, and have a certain predictive effect on HBV-related diseases.

Example 2:

1. Collection, processing and extraction of whole genome DNA from blood samples.

(1) Blood sample collection: Collect about 5ml of peripheral blood from HBV-related patients, put it into an anticoagulant tube containing EDTA, and let it stand for about 1 hour.

(2) Blood sample processing: The collected peripheral blood sample is diluted twice with pre-cooled PBS, slowly added to the lymphocyte separation medium of 2 times the sample volume, centrifuged at 2000rpm for 30min, and the middle milky white layer is divided into EP tubes (as shown in Figure 4, the circles represent lymphocytes), proceed to the next step.

(3) Extract whole-genome DNA: take the lymphocytes obtained in step (2), and extract whole-genome DNA according to D70K PureGene DNA isolation kits purchased from Qiagen.

2. Primer design and screening

(1) Primer design: use NCBI to check the full-length sequences of OR4F3 (NC_000005.10) and OR4F17 (NC_000019.10), and use Primer Premier 5 software to design 2 pairs of upstream and downstream primers for PCR for the full-length sequences (as shown in Table 1. shown), the primers were synthesized by Shenggong Company.

Table 1: 2 pairs of primers designed to detect OR4F3 and OR4F17 in the blood genome

name sequence R-H-OR4F3-DNA-F CCAGCTCCTCCTCCTAGTGT R-H-OR4F3-DNA-R CATTCTCCACACCACCAACGA R-H-OR4F17-DNA-F TGGTGTGCTCACTGTGTGTT R-H-OR4F17-DNA-R TGGACAGAGCTTTGGACGAC

Table 2: qPCR recipe for

Element volume SYBRgreen PCR Master Mix 10ul DNA template 0.5ul Primers (20mM each) 0.5ul water 9ul

Table 3: Quantitative PCR reaction conditions are

3. Difference detection of OR4F3 and OR4F17 gene copy numbers in blood genomes of normal people and HBV-related patients.

According to the above method, the gene copy numbers of OR4F3 and OR4F17 genes in 20 cases of normal human blood genome samples and 100 cases of CHB, 100 cases of LC and 100 cases of HBV-HCC were detected with the screened primers, and the results were found to be the same as those in 20 cases. Compared with normal samples, the copy numbers of OR4F3 and OR4F17 genes in 100 samples of CHB patients were significantly increased, and the copy numbers of OR4F3 and OR4F17 genes in 100 cases of LC and 100 samples of HBV-HCC patients were also increased, but lower than CHB patient specimens (see Figure 5).

The results suggest that OR4F3 and OR4F17 can be used as diagnostic markers for HBV-related diseases; OR4F3 and OR4F17 may become necessary and beneficial supplements of AFP in the diagnosis of liver cancer.

Four, with the correlation of the detection result of test kit of the present invention and HBV related disease as follows:

Extract the total DNA in the peripheral blood genome of the sample, and use the kit to first detect the copy number of OR4F3. Among them, when the copy number of OR4F3 in the blood genome is <15.66/ml, the sample has a 99.94% possibility of being normal; when the copy number of OR4F3 in the blood genome is >56.08/ml, the sample has a 93.21% possibility of being normal Hepatitis; when the copy number of OR4F3 in the blood genome is >56.08/ml<15.66/ml, the sample has a 49.43% probability of hepatitis and a 50.67% probability of liver cancer. For samples whose copy number of OR4F3 is between 15.66/ml and 56.08/ml, the copy number of OR4F17 is determined. When the copy number of OR4F17 is less than 14.28/ml, the sample has a 77.92% possibility of being liver cancer, and a 22.08% probability of being liver cancer. The possibility is hepatitis; when the copy number of OR4F17>14.28/ml, the sample has a 98.18% possibility of hepatitis (see Figure 2).

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. Modifications or replacements, these equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

Claims (4)

1.OR4F3 and applications of the OR4F17 in HBV relevant diseases diagnostic reagent or diagnostic kit is prepared；The HBV phases Related disorders are hepatitis B hepatitis, hepatic sclerosis or liver cancer；The diagnostic reagent is detection OR4F3 and the reagent of OR4F17 copy numbers.

2. OR4F3 according to claim 1 and OR4F17 is in HBV relevant diseases diagnostic reagent or diagnostic kit is prepared Application, it is characterised in that the reagent of the detection OR4F3 and OR4F17 copy numbers, are selected from：Have to OR4F3, OR4F17 Detect specific PCR primer.

3. OR4F3 according to claim 2 and OR4F17 is in HBV relevant diseases diagnostic reagent or diagnostic kit is prepared Application, it is characterised in that it is described have to OR4F3 and OR4F17 detect specific PCR primer such as SEQ ID NO:1~4 It is shown.

4. the OR4F3 and OR4F17 according to claim 1,2 or 3 are preparing HBV relevant diseases diagnostic reagent or diagnosis examination Application in agent box, it is characterised in that the biological sample that the diagnostic reagent or diagnostic kit are used to detect is peripheral blood.