Primer and probe composition, kit, and use in detection of EMX1 and CHFR gene methylation

Abstract

Provided are a primer and probe composition, a kit, and the use in the detection of EMX1 and CHFR gene methylation. The primer and probe composition comprises a primer pair and a probe set, wherein the primer pair comprises nucleotide sequences as shown in SEQ ID NOs: 4-5, SEQ ID NOs: 7-8 and SEQ ID NOs: 10-11; and the probe set comprises nucleotide sequences as shown in SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 12. The primers and probe set are designed at CpG sites near a target gene or a promoter of the target gene, can specifically bind to a methylated sequence, and can be used for detecting EMX1 and CHFR gene methylation to achieve highly sensitive, highly specific, accurate and rapid detection, and is thus suitable for auxiliary diagnosis of hepatocellular carcinoma.

Description

Primer and probe composition, kit and application in detecting EMX1, CHFR gene methylation TECHNICAL FIELD

[0001] The present application relates to the field of in vitro nucleic acid detection, in particular to primer and probe composition, kit and application in detecting EMX1, CHFR gene methylation. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is one of the most common human tumors. HCC can be divided into two major categories: primary and metastatic. Primary HCC mainly includes three different pathological types: hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA), and mixed hepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA). Hepatocellular carcinoma (HCC) accounts for about 75% of primary HCC.

[0003] Early diagnosis of HCC is crucial for early intervention and survival. Multiple studies have shown that early monitoring can improve overall survival, and international guidelines recommend that patients with cirrhosis should undergo HCC screening, as patients with cirrhosis have a 2-4% annual risk of developing HCC. The American Association for the Study of Liver Diseases (AASLD) classifies the following patients as high-risk patients for developing HCC: patients with cirrhosis (Child Pugh A and B), patients with cirrhosis (Child Pugh C waiting for liver transplantation), and patients without cirrhosis but with HBV.

[0004] The AASLD (American Association for the Study of Liver Diseases) currently recommends that high-risk patients receive abdominal ultrasound monitoring every six months. However, less than one-fifth of high-risk patients receive HCC screening. In addition, the sensitivity of abdominal ultrasound detection for early HCC is only 45%, and additional screening using the AFP biomarker may improve the detection rate of HCC. AFP is a serum glycoprotein whose elevation is associated with liver malignancies, and even with a combined approach, the sensitivity of detecting HCC is about 63%. The overall sensitivity of magnetic resonance imaging (MRI) and computed tomography (CT) for diagnosing HCC is 65% and 72%, respectively. However, for HCC smaller than 2 cm, the sensitivity of CT and MRI is only 48% and 62%, respectively, and for HCC smaller than 1 cm, the sensitivity of CT and MRI is even lower.

[0005] Liver biopsy remains a reliable diagnostic tool for HCC, and percutaneous, ultrasound-guided liver biopsy is the most commonly used method. The main disadvantage of tissue biopsy is its invasiveness; the most important risk is bleeding. Therefore, the clinical diagnostic needs for liver cancer are far from being met. Based on the concept of precision medicine, developing new blood-based diagnostics is a key strategy to improve early diagnosis of HCC.

[0006] A large number of candidate markers are currently being studied, of which cfDNA methylation is a promising marker. DNA methylation is a form of DNA chemical modification, in which a methyl group is covalently bound to the 5th carbon of cytosine in the CpG dinucleotide of the genome; it can affect expression, gene and protein interaction without changing the DNA sequence; generally, cfDNA is derived from dead cells, such as white blood cells, but in cancer patients, a small amount of cfDNA can also be released into the circulation by tumor cells due to necrosis or apoptosis. This circulating tumor-derived DNA (ctDNA) can be analyzed to understand the tumor-specific gene methylation pattern. Studies have shown that abnormal methylation of certain tumor-related genes can occur as early as the early stages of tumor development, and can be used as a specific marker for diagnosis and monitoring. In fact, comparison of the whole genome profile in cfDNA reveals high-methylation or low-methylation gene markers specific to tumors (referred to as DNA methylation markers (DMM)), thus enabling accurate differentiation between early HCC and cirrhosis.

[0007] Among the many methylation detection methods, real-time fluorescent quantitative PCR, which is efficient, fast and easy to operate, is one of the main methods. Currently, there is a lack of accurate non-invasive auxiliary diagnostic techniques for liver cancer in the first line of clinical treatment, and it is necessary to develop products based on gene methylation detection for auxiliary diagnosis of liver cancer. SUMMARY

[0008] The purpose of the present application is to provide primer and probe compositions, kits and applications in detecting EMX1 and CHFR gene methylation, to achieve high sensitivity, high specificity, accurate and rapid detection suitable for auxiliary diagnosis of hepatocellular carcinoma.

[0009] In the previous site screening work, we detected the RRBS methylation sites of 224 liver cancer clinical samples and 276 normal human samples, and referred to public databases, and from millions of methylation sites, we optimized 10 differential methylation targets (DMR). However, due to the large number of sites, the cost and practicability of the system are poor, therefore, we selected 5 target sites from them, and through qPCR detection of an additional 122 samples (including 50 patients and 72 healthy people), we finally selected 2 target sites and determined the appropriate threshold.

[0010] It should be pointed out that the design of any primer cannot achieve high sensitivity and specificity detection, for two reasons: first, the same gene CpG site can be as many as several thousand, and the detection efficiency of different CpG sites is different; second, the detection efficiency of different genes is different, and by increasing the number of genes, the sensitivity and specificity can be improved to a certain extent, but the increase in the number of genes will greatly reduce the practicability, the present application is based on the methylation site of the genome, the best CpG site and the best combination of genes are selected to achieve high sensitivity and specificity, which meets the clinical needs while taking into account the better applicability.

[0011] Therefore, the present application provides the following solutions: In a first aspect, the present application provides a primer and probe composition for detecting EMX1 and CHFR gene methylation, which comprises a primer pair and a probe set, the primer pair comprises nucleotide sequences as shown in SEQ ID NO: 4-5, SEQ ID NO: 7-8, and SEQ ID NO: 10-11; the probe set comprises nucleotide sequences as shown in SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO: 12; further, the 5' end of the probe set is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group; preferably, the fluorescent groups are different and are selected from FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red or CY3; the quenching groups are independently selected from MGB, BHQ-1, BHQ-2, BHQ-3 or TAMRA; preferably, the 5' end of the probe sequence as shown in SEQ ID NO: 6 is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ-1 fluorescent quenching group; the 5' end of the probe sequence as shown in SEQ ID NO: 9 is labeled with a VIC fluorescent reporter group, and the 3' end is labeled with a BHQ-1 fluorescent quenching group; the 5' end of the probe sequence as shown in SEQ ID NO: 12 is labeled with a CY5 fluorescent reporter group, and the 3' end is labeled with a BHQ-3 fluorescent quenching group; In a second aspect, the present application provides an EMX1 and CHFR gene methylation detection kit, which comprises the primer and probe composition described in the first aspect. The kit provided by the present application uses the TaqMan probe multiplex fluorescence PCR method, the primer probe is designed near the CpG site of the target gene or the target gene promoter, can specifically bind to the methylated sequence and undergo PCR reaction, while the unmethylated sequence cannot bind to it. In addition, the transformed internal reference gene ACTB is detected at the same time to evaluate whether the DNA template is qualified.

[0012] The working principle of Taqman probe method is as follows: the 5' end of the probe is labeled with a fluorescent reporter group (such as FAM), and the 3' end is labeled with a fluorescent quenching group, and the two constitute an energy transfer structure. Therefore, when the probe is intact, the fluorescence signal excited by the 5' end fluorescent reporter group is absorbed or inhibited by the 3' end quenching group, and no fluorescence signal change occurs. When there is a target gene in the PCR reaction system, specific nucleic acid fragments will be amplified, and the fluorescent probe will hybridize according to the principle of base pairing. When PCR enters the extension (replication) period, Taq enzyme starts from the 3' end of the primer and extends along the DNA template. When it extends to the position of the probe, the 5'→3' exonuclease activity will cut the probe. At this time, the energy transfer structure between the fluorescent reporter group and the quenching group is destroyed, the quenching effect of the quenching group is released, and the fluorescence signal of the fluorescent reporter group is released. With the accumulation of fluorescence signal, it can be detected by fluorescence PCR instrument. Relatively, when the probe sequence does not match the target gene sequence, the probe cannot bind to the target sequence, and the specific probe cannot be cut during the PCR extension process, and the corresponding fluorescence signal will not appear.

[0013] Further, the kit further comprises a positive control, a negative control, a PCR reaction mixture and purified water.

[0014] Preferably, the positive control is an EMX1, CHFR methylation gene sequence plasmid, and the negative control is DNA transformed from a normal genome; and / or, the PCR reaction mixture comprises DNA polymerase, dNTP, Mg 2+ , auxiliary enhancer and buffer.

[0015] In a third aspect of the present application, the kit of the second aspect is used for detecting EMX1, CHFR gene methylation for non-diagnostic purposes. Non-diagnostic purposes include detection of samples that cannot be traced back to humans, such as detection of gene methylation of a sample under laboratory conditions. The detection result only represents the result of the sample itself, i.e. the presence or absence of EMX1, CHFR gene methylation.

[0016] In a fourth aspect of the present application, a method for detecting EMX1, CHFR gene methylation for non-diagnostic purposes is provided, comprising the following steps: extracting genomic DNA from the sample to be tested and performing bisulfite transformation; using the transformed DNA as a template, performing fluorescent quantitative PCR reaction using the kit of the second aspect; and determining the methylation state of the gene in the sample to be tested according to the fluorescent quantitative PCR detection result.

[0017] Further, the sample to be detected is blood, serum or plasma.

[0018] Compared with the prior art, the application has the following beneficial effects: the primers and probe set provided by the application are designed at the CpG site near the target gene or the promoter of the target gene, can specifically bind to the methylated sequence, and can realize high sensitivity, high specificity, accurate and rapid detection of EMX1 and CHFR gene methylation, so as to be suitable for auxiliary diagnosis of hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of the drawings.

[0020] Fig. 1 is a design diagram of the EMX1 methylation site primer probe according to the application.

[0021] Fig. 2 is a design diagram of the CHFR methylation site primer probe according to the application.

[0022] Fig. 3 is a design diagram of the ACTB methylation site primer probe according to the application. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and beneficial technical effects of the application more clear, the application will be further described in detail in combination with the drawings and specific embodiments. It should be understood that the specific embodiments described in the specification are only for the purpose of explaining the application, and are not intended to limit the application.

[0024] Example 1: Design of the kit DNA methylation is a form of DNA chemical modification, that is, a methyl group is covalently combined with the 5th carbon atom of cytosine on the CpG dinucleotide to form 5-methylcytosine. In order to detect the CpG cytosine methylation of DNA, the DNA needs to be treated by bisulfite conversion, so that the unmethylated cytosine (C) is converted into uracil (U), while the methylated cytosine will not be converted, thereby specifically distinguishing the methylated and unmethylated target genes.

[0025] The transformed wild-type EMX1 target sequence, CHFR target sequence, and ACTB target sequence are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The primers or probes are designed according to the gene site shown in FIGS. 1-3. The primer-probe combination of the above-mentioned EMX1 and CHFR genes avoids mutual interference between multiple primer pairs and corresponding detection probes through the ingenious design of the amplification primer pair and the detection probe. It has been verified that the use of the above-mentioned primer-probe combination for detecting EMX1 and CHFR gene methylation has good specificity and high sensitivity.

[0026] Specifically, the PCR amplification primers and the fluorescein-modified characteristics of the EMX1 and CHFR methylation genes and the reference site are shown in Table 1.

[0027] Table 1: PCR amplification primer pairs, probe sets, and fluorescein labeling characteristics of three sites

[0028] Specifically, the detection kit further comprises a PCR amplification system for detecting EMX1 and CHFR gene methylation, which comprises at least one of DNA polymerase, dNTP, Mg 2+ , PCR buffer system, PCR enhancer, and PCR stabilizer, including Tris, KCl (10 mM-500 mM), (NH4)2SO4(10 mM-500 mM), glycerol (1 %-50 %), BSA (0.001 mg / mL-1 mg / mL), Tween 20 (0.1 %-10 %), dithiothreitol (1 mM / mL-100 mM / mL), betaine (0.1 M-3 M), and DMSO (0.1 %-10 %). The summary is shown in Table 2.

[0029] Table 2: Components of the liver cancer EMX1 and CHFR methylation detection kit

[0030] Example 2: Detection process and results 1. Sample pretreatment: Before detection by the kit, sample DNA extraction and transformation are required. The plasma DNA extraction and sulfite transformation kit (kit name: QIAamp Circulating Nucleic Acid Kit; item number: 55114; EZ DNA Methylation-Lightning Kit; company name: ZYMO RESEARCH;) is used for sample pretreatment. The volume of the plasma sample required by the kit should be greater than 2 ml, and the pretreatment is performed according to the requirements of the plasma DNA extraction and sulfite transformation kit instructions. The treated plasma DNA can be directly used for detection by the kit.

[0031] 2. Sample detection: 1) Take out the kit stored at -15 to -25℃ in the dark, and equilibrate the kit components to room temperature; 2) Take out the corresponding number of 96-well plates or PCR reaction tubes; 3) Calculate and remove the corresponding amount of reagents according to the number of reactions performed in the experiment, which refers to the number of test samples plus 1 blank control (using purified water instead of DNA), 1 mutant control, and 1 normal control, and the remaining reagents are stored at -15 to -25℃ in the dark; 4) Prepare the reaction according to Table 3.

[0032] 5) Template solution refers to the sample mixture solution after pretreatment in 1, methylated positive quality control, methylated negative quality control, and blank control; 6) Seal the 96-well plate or cover the PCR reaction tube, shake uniformly, centrifuge at 2000 rpm for 10 seconds, and place it in a fluorescent quantitative PCR instrument; 7) Perform PCR amplification according to the conditions in Table 4.

[0033] Table 4: Amplification program

[0034] 3. Result analysis: Refer to the instrument user manual for settings. The following analysis takes ABI series instruments as an example: 1) Adjust the fluorescence signal threshold to the target fluorescence channel without signal for the methylated negative quality control and with detection signal for ACTB in the methylated control detection results. For example, using ABI 7500 fluorescent quantitative PCR instrument, set the baseline to 10-22 cycles and the fluorescence signal threshold to about 25000 to obtain ideal detection results; 2) The fluorescence channel corresponding to the detected target gene in the detection results of this kit is shown in Table 5.

[0035] Table 5:

[0036] 4. Quality control 1) Exogenous control: This kit contains positive and negative control samples. These controls should be added in each reaction to detect whether the reaction is successful and ensure the effectiveness of the reaction. The reaction Ct value of the positive and negative control samples should be within the effective range, as shown in Table 6. If the reaction Ct value is outside the effective range, the test results are invalid.

[0037] Table 6:

[0038] 2) Endogenous control: Endogenous control can detect the ACTB gene converted by sulfite, and can also detect whether the sample meets the experimental requirements (including whether the sample preparation is correct and whether the sample DNA content meets the requirements). If the Ct value of ACTB exceeds the effective range, the reaction is invalid. Because a too high Ct value indicates that the BisDNA concentration is too low or the PCR reaction is inhibited.

[0039] 3) Blank control: no signal in the detection channel Ct value in the detection result. If the signal rises and the Ct value is ≤ 34, the experimental result is invalid, and it is recommended to retest.

[0040] 5. Result interpretation The PCR result is shown in Table 7.

[0041] Table 7:

[0042] *: The difference between the Ct value of the target gene (EMX1 and CHFR) and the Ct value of ACTB. (The positive and negative of the PCR result is determined by the Ct value, not by the S-shaped curve)

[0043] Table 8:

[0044] The sample information and detection results are shown in Table 9.

[0045] Table 9: According to the following formula, the combined sensitivity of EMX1 and CHFR genes reached 84%, and the specificity reached 92%, thus better compensating for the existing detection means, such as the low accuracy of conventional AFP means, which has important significance for timely detection of early cancer lesions.

[0046] The present application is not limited to the description and embodiments described in the specification, and therefore additional advantages and modifications can be easily realized by those skilled in the art, and the present application is not limited to specific details, representative devices and examples of the drawings shown and described herein. The spirit and scope of the general concept defined by the claims and their equivalents.

Claims

1. A primer and probe composition for detecting EMX1, CHFR gene methylation, characterized in that, The primer pair comprises nucleotide sequences as shown in SEQ ID NO: 4-5, SEQ ID NO: 7-8, SEQ ID NO: 10-11; and the probe set comprises nucleotide sequences as shown in SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO:

12.

2. The primer and probe composition of claim 1, wherein, The probe set is labeled with a fluorescent group at the 5' end and a fluorescent quenching group at the 3' end.

3. The primer and probe composition of claim 2, wherein, The fluorescent group is selected from FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red or CY3, and the quenching group is independently selected from MGB, BHQ1, BHQ2, BHQ3 or TAMRA.

4. The primer and probe composition of claim 2, wherein, The probe sequence as shown in SEQ ID NO: 6 is labeled with a FAM fluorescent reporter group at the 5' end and a BHQ1 fluorescent quenching group at the 3' end; the probe sequence as shown in SEQ ID NO: 9 is labeled with a VIC fluorescent reporter group at the 5' end and a BHQ1 fluorescent quenching group at the 3' end; and the probe sequence as shown in SEQ ID NO: 12 is labeled with a CY5 fluorescent reporter group at the 5' end and a BHQ3 fluorescent quenching group at the 3' end.

5. An EMXl, CHFR gene methylation detection kit, characterized by, The kit further comprises a positive control, a negative control, a PCR reaction mixture and purified water.

6. The kit of claim 5, wherein 8. Use of the kit of any one of claims 5-7 for detecting methylation of EMX1 and CHFR genes for non-diagnostic purposes.

7. The kit of claim 6, wherein The positive control is a plasmid of EMX1, CHFR methylation gene sequence, the negative control is DNA after normal genome transformation; and / or, the PCR reaction mixture contains DNA polymerase, dNTP, Mg 2+ , auxiliary enhancer and buffer. The kit comprises:

9. A method for detecting methylation of EMX1, CHFR gene for non-diagnostic purposes, characterized by the steps of extracting genomic DNA from the sample to be tested and performing bisulfite conversion; using the kit of any one of claims 5-7 to perform a fluorescent quantitative PCR reaction using the converted DNA as a template; judging the methylation state of the gene in the sample to be tested according to the results of the fluorescent quantitative PCR detection. The sample to be tested is blood, serum or plasma.

10. The method of claim 9, wherein, ​

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