Method and kit for detecting KCNJ5 gene mutation

Abstract

The invention is applicable to the technical field of biomedicine, and provides a method and a kit for detecting KCNJ5 gene mutation, and the method comprises the following steps: extracting a cfDNA sample; the method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification reaction by using a specific primer and a probe aiming at a KCNJ5 gene mutation site and taking a sample as a template; the specific primers and probes aiming at the mutation site of the KCNJ5 gene comprise specific primers and probes aiming at the mutation site of the G151R (c.451Ggt; the invention relates to a specific primer and a probe for G151R (c.451Ggt; the invention relates to a specific primer and a probe of L168R (c.503Tgt; C), aiming at L168R (c.503Tgt; g), a specific primer and a probe; sequencing a PCR amplification product, analyzing a sequencing result, and determining whether the KCNJ5 gene mutation exists or not. According to the invention, high-sensitivity and high-specificity detection of KCNJ5 gene mutation is realized by combining the specific primer and the probe with a digital PCR technology, and three main mutation types of the KCNJ5 gene can be detected at 0.01% mutation allele frequency level.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method and a kit for detecting KCNJ5 gene mutations. Background Art

[0002] Primary aldosteronism (PA) is a common cause of secondary hypertension. Aldosterone-producing adenoma (APA) is one of the main causes of PA. 40-65% of APA patients have somatic mutations, among which KCNJ5 gene mutations are the most common. The mutation types of the KCNJ5 gene mainly include p.Gly151Arg (G151R) and p.Leu168Arg (L168R). At present, the detection of KCNJ5 gene mutations mainly relies on postoperative tumor tissue biopsy and cannot perform gene typing evaluation before surgery. Therefore, there is an urgent need for a non-invasive liquid biopsy method that can be used for early screening and auxiliary diagnosis of PA patients.

[0003] Although the existing ARMS technology can detect DNA point mutations, the early technology was limited by the specificity of DNA polymerase and required multiple optimizations. The third-generation ARMS technology (Perfect-ARMS) combines a DNA polymerase with ultra-high specificity (Shenzhen SNUPP polymerase), which can accurately identify single-base mismatches, significantly reduce non-specific amplification. Digital PCR (dPCR) technology divides the sample into tens of thousands of independent reaction units to achieve absolute counting of target molecules, with extremely low detection limits and high specificity, especially suitable for the detection of cell-free DNA (cfDNA) in plasma. Summary of the Invention

[0004] An object of an embodiment of the present invention is to provide a method for detecting KCNJ5 gene mutations, aiming to solve the problems raised in the above background art.

[0005] An embodiment of the present invention is implemented as follows. A method for detecting KCNJ5 gene mutations includes the following steps:

[0006] (1) Extract cfDNA samples;

[0007] (2) Using specific primers and probes targeting the KCNJ5 gene mutation sites, with the sample as a template, perform a PCR amplification reaction; there are three types of specific primers and probes targeting the KCNJ5 gene mutation sites, including specific primers and probes for G151R (c.451G>A), the sequences of the primer pair are as shown in SEQ ID NO:1-2, and the sequence of the probe is as shown in SEQ ID NO:3; specific primers and probes for G151R (c.451G>C), the sequences of the primer pair are as shown in SEQ ID NO:4-5, and the sequence of the probe is as shown in SEQ ID NO:6; specific primers and probes for L168R (c.503T>G), the sequences of the primer pair are as shown in SEQ ID NO:7-8, and the sequence of the probe is as shown in SEQ ID NO:9;

[0008] (3) Sequence the PCR amplification products, analyze the sequencing results, and determine whether there is a KCNJ5 gene mutation.

[0009] Preferably, in step (2), the reaction program of the PCR amplification reaction is: 58°C for 2 min; 95°C for 2 min; then perform 8 cycles, each cycle including 95°C for 30 s, 60°C for 1 min, and then perform 45 cycles, each cycle including 95°C for 30 s, 58°C for 1 min.

[0010] Another object of the embodiments of the present invention is to provide a kit for detecting KCNJ5 gene mutations, and the kit includes specific primers and probes targeting the KCNJ5 gene mutation sites;

[0011] There are three types of specific primers and probes targeting the KCNJ5 gene mutation sites, including specific primers and probes for G151R (c.451G>A), the sequences of the primer pair are as shown in SEQ ID NO:1-2, and the sequence of the probe is as shown in SEQ ID NO:3; specific primers and probes for G151R (c.451G>C), the sequences of the primer pair are as shown in SEQ ID NO:4-5, and the sequence of the probe is as shown in SEQ ID NO:6; specific primers and probes for L168R (c.503T>G), the sequences of the primer pair are as shown in SEQ ID NO:7-8, and the sequence of the probe is as shown in SEQ ID NO:9.

[0012] Preferably, both ends of the probe are respectively provided with a fluorescent group and a fluorescence quenching group.

[0013] Preferably, the kit further includes primers and probes for the wild sequence of KCNJ5, the sequences of the primer pair are as shown in SEQ ID NO:10-11, and the sequence of the probe is as shown in SEQ ID NO:12.

[0014] Preferably, both ends of the probe are respectively provided with a fluorescent group and a fluorescence quenching group.

[0015] A method for detecting KCNJ5 gene mutations provided by an embodiment of the present invention uses specific primers and probes in combination with digital PCR technology to achieve highly sensitive and highly specific detection of KCNJ5 gene mutations. This method can detect three main mutation types (G151R c.451G>C, G151R c.451G>A, L168R c.503T>G) of the KCNJ5 gene at a mutant allele frequency (VAF) level of 0.01%, and calculate the proportion of mutant copies. Compared with traditional detection methods, the embodiment of the present invention has the characteristics of non-invasiveness (detecting plasma cfDNA through liquid biopsy, avoiding the trauma of traditional tissue biopsy), high sensitivity, high specificity (able to detect KCNJ5 gene mutations at a 0.01% VAF level), high degree of automation (using a fully automatic integrated chip-type digital PCR instrument, with simple operation and easy clinical promotion), and easy clinical promotion. It is particularly suitable for the early screening and auxiliary diagnosis of primary aldosteronism (PA), providing a new technical means for the precision medicine of PA patients. Description of the Drawings

[0016] Figure 1 It is the primer specificity detection result provided by the embodiment of the present invention;

[0017] Figure 2 It is the primer sensitivity detection result provided by the embodiment of the present invention;

[0018] Figure 3 It is the two-dimensional scatter plot result of the clinical sample detection provided by the embodiment of the present invention. Detailed Embodiments

[0019] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] The following describes the specific implementation of the present invention in detail with reference to specific embodiments.

[0021] Embodiment 1. A method for detecting KCNJ5 gene mutations includes the following steps:

[0022] (1) Collect 5 mL of venous EDTA anticoagulated blood, centrifuge at 3000 rpm for 10 min to separate plasma, and use a commercial cfDNA extraction kit to extract free nuclei (cfDNA) from the plasma;

[0023] (2) An amplification detection all-in-one microfluidic chip digital PCR instrument is used, combined with the Perfect-ARMS technology. Specific primers and probes targeting the KCNJ5 gene mutation site are used for detection. The sequences of the specific primers and probes are shown in Table 1, and the digital PCR reaction system is shown in Table 2. The reaction program is 58 °C for 1 min; 95 °C for 2 min; then 8 cycles are carried out, each cycle including 95 °C for 30 s and 60 °C for 1 min, and then 45 cycles are carried out, each cycle including 95 °C for 30 s and 58 °C for 1 min:

[0024] Table 1

[0025]

[0026]

[0027]

[0028] Table 2

[0029] (3) By statistically analyzing the fluorescence signals of each reaction unit, the copy number of the KCNJ5 gene mutation and the wild-type copy number are calculated, and then the VAF is calculated. The VAF = (mutation copy number / total copy number) × 100%;

[0030] Among them, the primer specificity detection results are as Figure 1 shown, and the primer sensitivity detection is as Figure 2 shown. It can be seen that the specificity is 100%, and the sensitivity can detect the VAF level of 0.01%.

[0031] Using this detection method for clinical sample verification, the specific steps are as follows:

[0032] (1) Collect tumor tissue and plasma samples from 20 patients with aldosterone adenoma;

[0033] (2) Extract tumor tissue nucleic acid and plasma cfDNA respectively, and use the above detection method for detection;

[0034] (2) Use the detection result of the tumor tissue sample as the positive control and the normal tissue sample as the negative control;

[0035] (4) Obtain the PCR scatter plot as Figure 3 shown. The results show that the KCNJ5 gene mutation was detected in the tumor tissues of 11 patients, and the mutation detection frequency was 55%; among them, the mutation was detected in the plasma cfDNA of 6 patients, and the mutation detection frequency was 55%.

[0036] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting KCNJ5 gene mutation, characterized in that: The following steps are involved: (1) Extract cfDNA samples; (2) using specific primers and probes for the mutation site of the KCNJ5 gene and taking the sample as a template, a PCR amplification reaction is performed; the specific primers and probes for the mutation site of the KCNJ5 gene are of three types, including specific primers and probes for G151R (c.451G>A), the sequences of the primer pairs are shown in SEQ ID NOs: 1-2, and the sequences of the probes are shown in SEQ ID NOs: 3; specific primers and probes for G151R (c.451G>C), the sequences of the primer pairs are shown in SEQ ID NOs: 4-5, and the sequences of the probes are shown in SEQ ID NOs: 6; specific primers and probes for L168R (c.503T>G), the sequences of the primer pairs are shown in SEQ ID NOs: 7-8, and the sequences of the probes are shown in SEQ ID NOs: 9; (3) Sequence the PCR amplification products and analyze the sequencing results to determine whether the KCNJ5 gene mutation exists.

2. The method for detecting KCNJ5 gene mutation according to claim 1, characterized in that: In step (2), the reaction procedure of the PCR amplification reaction is: 58°C for 2 min; 95°C for 2 min; followed by 8 cycles, each cycle including 95°C for 30 s and 60°C for 1 min, followed by 45 cycles, each cycle including 95°C for 30 s and 58°C for 1 min.

3. A kit for detecting KCNJ5 gene mutation, characterized in that: The kit includes specific primers and probes for the mutation site of KCNJ5 gene; There are three types of specific primers and probes for the KCNJ5 gene mutation site, including specific primers and probes for G151R (c.451G>A), the sequence of the primer pair is shown in SEQ ID NO: 1-2, and the sequence of the probe is shown in SEQ ID NO: 3; specific primers and probes for G151R (c.451G>C), the sequence of the primer pair is shown in SEQ ID NO: 4-5, and the sequence of the probe is shown in SEQ ID NO: 6; specific primers and probes for L168R (c.503T>G), the sequence of the primer pair is shown in SEQ ID NO: 7-8, and the sequence of the probe is shown in SEQ ID NO:

9.

4. The kit for detecting KCNJ5 gene mutation according to claim 3, characterized in that: The two ends of the probe are respectively provided with a fluorescent group and a fluorescent quenching group.

5. The kit for detecting KCNJ5 gene mutation according to claim 3, characterized in that: The kit also includes primers and probes of KCNJ5 wild sequence, the sequences of the primer pairs are shown in SEQ ID NO:10-11, and the sequence of the probe is shown in SEQ ID NO:

12.

6. The method for detecting KCNJ5 gene mutation according to claim 5, characterized in that: The two ends of the probe are respectively provided with a fluorescent group and a fluorescent quenching group.

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