A primary aldosteronism subtype diagnosis kit based on plasma exosome KCNJ5 gene mutation detection and application thereof

Abstract

The application relates to a primary aldosterone hyperplasia subtype diagnosis kit based on plasma exosome KCNJ5 gene mutation detection and application thereof, wherein the kit comprises an exosome enrichment component, an exosome lysis reagent and a nucleic acid detection reagent; the exosome enrichment component comprises enrichment reagents matched with a high-gradient magnetic separation device; the exosome lysis component comprises a lysis solution for extracting exosome nucleic acids; the nucleic acid detection reagent is used for detecting KCNJ5 gene mutation sites, and the mutation sites comprise one or more of G151R (c.451G>A), G151R (c.451G>C) and L168R (c.503T>G). The kit integrates the high-gradient magnetic separation component, the exosome lysis reagent and the digital PCR detection reagent, realizes the full-process standardization from sample treatment to result interpretation, is clear in operation steps, low in technical requirements for personnel, short in detection period, and can be rapidly developed in medical institutions with digital PCR instruments.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a diagnostic kit for primary aldosteronism subtypes based on the detection of KCNJ5 gene mutations in plasma exosomes and its application. Background Technology

[0002] Primary aldosteronism (PA) is the most common secondary hypertension, caused by excessive aldosterone secretion from the adrenal cortex. Clinically, it mainly manifests as hypertension and hypokalemia. Compared to patients with primary hypertension, PA patients experience more severe damage to target organs such as the heart, brain, and kidneys. Therefore, early diagnosis and precise treatment are crucial for improving patient prognosis. Epidemiological studies show that the prevalence of PA exceeds 10% in the general hypertensive population, and this proportion is as high as 17%–23% in patients with refractory hypertension. However, the current screening rate for PA in the hypertensive population is less than 1%, highlighting the urgent clinical need to develop simple, easy-to-use screening methods with both high sensitivity and specificity.

[0003] Primary aldosteronism can be classified into several subtypes based on its etiology and pathophysiological characteristics, mainly including: aldosterone-producing adenoma (APA), idiopathic hyperaldosteronism (IHA), primary adrenal hyperplasia, familial aldosteronism, aldosterone-secreting adrenocortical carcinoma, and ectopic aldosterone-secreting tumors. Among these, the APA subtype accounts for approximately 35% of primary aldosteronism patients, and the IHA subtype accounts for approximately 60%. Clinical practice shows that APA patients can achieve complete cure of hypertension through unilateral adrenalectomy, significantly reducing the long-term risk of cardiovascular events. Therefore, accurate diagnosis of the APA subtype is crucial for developing a reasonable treatment plan (surgical or pharmacological).

[0004] However, the classification and diagnosis of primary aldosteronism has always been a key focus and challenge in clinical practice. Currently, commonly used diagnostic and classification methods mainly include:

[0005] 1) Aldosterone-renin Ratio (ARR) Screening: ARR is the preferred screening indicator for primary aldosteronism, and an elevated ARR suggests the possibility of primary aldosteronism. However, the results of this test are easily affected by various factors, including the patient's antihypertensive medications and serum potassium levels. Its false positive rate can be as high as 30%, so it needs to be combined with serum potassium and renin levels for comprehensive judgment and is not suitable for precise subtyping.

[0006] 2) Adrenal CT scan: CT scans can provide a preliminary assessment of morphological changes in the adrenal glands, such as the discovery of unilateral adenomas, nodules, or hyperplasia. However, its diagnostic accuracy is limited (approximately 50%-70%), making it difficult to distinguish between non-functional and functional adenomas. Furthermore, it has insufficient ability to identify microadenomas or bilateral lesions, leading to a risk of missed diagnoses or misdiagnoses.

[0007] 3) Bilateral Adrenal Venous Sampling (AVS): Currently recognized as the gold standard for the classification and diagnosis of primary aldosteronism. Blood is collected from both adrenal veins via catheter insertion, and the aldosterone-to-cortisol ratio is measured to determine whether aldosterone hypersecretion is unilateral or bilateral. Its sensitivity and specificity can both exceed 90%. However, AVS is an invasive procedure requiring highly skilled physicians and carries risks of complications such as adrenal vein rupture and hematoma. Even in high-level medical centers, the failure rate of AVS (e.g., catheterization failure, sample dilution) remains as high as approximately 10%.

[0008] In recent years, the development of liquid biopsy technology has provided new ideas for disease diagnosis. Our prior application CN120210356A discloses a method and kit for detecting KCNJ5 gene mutations. However, this technology has the following inherent drawbacks: It uses plasma cell-derived DNA (cfDNA) as the detection target carrier. Because cfDNA is in a free state, it is easily degraded by nucleases in plasma, requiring extremely high standards for sample processing and preservation, making it difficult to promote in primary hospitals and unable to meet the needs of large-scale screening. Furthermore, cfDNA originates from apoptosis release of various tissues and cells throughout the body, resulting in highly mixed sources and making it difficult to specifically reflect the gene status of adrenal lesions. Even if a KCNJ5 mutation is detected, it is impossible to determine whether the mutation originates from adrenal lesions or background interference from other tissues.

[0009] In summary, existing diagnostic methods for primary aldosteronism subtypes suffer from problems such as invasiveness, operational complexity, limited accuracy, and difficulty in large-scale implementation. In particular, while cfDNA-based detection methods can achieve generalized screening for primary aldosteronism, their poor sample stability and demanding operational requirements make them difficult to widely apply in clinical practice. Summary of the Invention

[0010] (a) Technical problems to be solved

[0011] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection and its application. It solves the technical problems of existing primary aldosteronism subtyping diagnostic methods, such as invasiveness, low accuracy, poor sample stability, high operational requirements, and difficulty in widespread application in clinical practice.

[0012] (II) Technical Solution

[0013] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0014] In a first aspect, embodiments of the present invention provide the application of plasma exosome KCNJ5 gene mutation in the preparation of diagnostic reagents for primary aldosteronism subtypes, characterized in that the mutation site of the KCNJ5 gene is selected from at least one of the following: G151R (c.451G>A), G151R (c.451G>C) and L168R (c.503T>G).

[0015] The diagnostic reagent is used to detect the mutation status of the KCNJ5 gene in plasma exosomes. When one or more of the mutation sites are detected as positive, it indicates that the subject is a high-risk group for the primary aldosteronism subtype.

[0016] The diagnostic reagent uses digital PCR technology to detect mutation sites in the KCNJ5 gene. The digital PCR technology uses LNA-modified specific probes. The reaction conditions for the digital PCR are: 55℃ reverse transcription for 20 min, 95℃ pre-denaturation for 2 min, 95℃ denaturation for 30 s, 56℃ annealing extension for 30 s, for a total of 45 cycles.

[0017] Secondly, embodiments of the present invention provide a diagnostic kit for primary aldosteronism subtypes based on the detection of KCNJ5 gene mutations in plasma exosomes, characterized in that the kit includes an exosome enrichment component, an exosome lysis reagent, and a nucleic acid detection reagent;

[0018] The exosome enrichment component includes enrichment reagents adapted to a high-gradient magnetic separation device; the exosome lysis component includes lysis buffer for extracting exosome nucleic acids; and the nucleic acid detection reagent is used to detect KCNJ5 gene mutation sites, the mutation sites including one or more of G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G).

[0019] The enrichment conditions of the high gradient magnetic separation device are as follows: plasma sample volume is 1 mL, plasma sample is injected into the high gradient magnetic separation device at a flow rate of 50-200 μL / min, magnetic separation time is 5-20 min, and separation temperature is 25℃.

[0020] As a preferred embodiment of the present invention, the nucleic acid detection reagent includes an LNA-modified specific probe for detecting KCNJ5 gene mutation sites, primer pairs, PCR reaction buffer, DNA polymerase, and dNTPs.

[0021] In a preferred embodiment of the present invention, the LNA-modified specific probes are designed with sequences targeting each mutation site as follows:

[0022] The probe targeting the G151R (c.451G>A) mutation site has a nucleotide sequence containing an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 1;

[0023] The probe targeting the G151R (c.451G>C) mutation site has a nucleotide sequence containing an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 2;

[0024] The probe targeting the L168R (c.503T>G) mutation site has a nucleotide sequence containing an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 3.

[0025] Among them, SEQ ID NO: 1 is the target sequence of the KCNJ5 gene G151R (c.451G>A) mutant, SEQ ID NO: 2 is the target sequence of the KCNJ5 gene G151R (c.451G>C) mutant, and SEQ ID NO: 3 is the target sequence of the KCNJ5 gene L168R (c.503T>G) mutant.

[0026] In a preferred embodiment of the present invention, the high gradient magnetic separation device includes a separation column having a flow cavity, and a magnet disposed around the separation column to generate a magnetic field.

[0027] A magnetic medium is provided inside the flow cavity. The magnetic medium can generate a high gradient magnetic field inside the flow cavity under the action of a magnetic field, so as to capture and enrich exosomes in the plasma sample.

[0028] In a preferred embodiment of the present invention, the magnetic medium is one or more of nickel mesh, iron mesh, and cobalt mesh.

[0029] As a preferred embodiment of the present invention, a permanent magnet is also included, which is configured to adsorb and fix the magnetic medium after lysis to separate the supernatant containing nucleic acid.

[0030] In a preferred embodiment of the present invention, the nucleic acid detection reagent is an amplification reagent suitable for a digital PCR instrument, and the reaction conditions of the digital PCR are: reverse transcription at 55°C for 20 min, pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, and annealing extension at 56°C for 30 s, for a total of 45 cycles.

[0031] In a preferred embodiment of the present invention, the digital PCR instrument is a digital droplet PCR instrument.

[0032] In a preferred embodiment of the present invention, the kit further includes calibrators and quality control samples, wherein the calibrators are standard plasmids containing specific mutation sites of the KCNJ5 gene, and the quality control samples are plasmids containing the wild-type KCNJ5 gene.

[0033] (III) Beneficial Effects

[0034] The beneficial effects of this invention are as follows: This invention provides a diagnostic kit for primary aldosteronism (APA) subtypes based on the detection of KCNJ5 gene mutations in plasma exosomes and its application. It provides a basis for diagnosing APA subtypes by detecting KCNJ5 gene mutations in peripheral blood exosomes. Using plasma exosomal DNA as the target carrier for APA subtype diagnosis, compared to existing technologies, the lipid bilayer membrane structure of exosomes effectively protects the internal DNA from nuclease degradation. Samples can be stably stored for a long time at -80℃ and are less prone to failure during transportation, solving the technical problems of stringent sample processing and storage conditions for cfDNA detection, which hinders its widespread adoption in primary hospitals. Simultaneously, exosomes are specifically secreted from diseased adrenal tissue, enriching target molecules from diseased tissue at the source, effectively eliminating background interference from normal tissue, significantly improving detection specificity, and laying the foundation for accurate subtyping.

[0035] This invention applies high-gradient magnetic separation technology to the enrichment and purification of plasma exosomes. Using ferromagnetic media such as nickel mesh in a high-gradient magnetic field, exosomes in plasma can be efficiently captured, increasing target concentration by 10-100 times. The operation is simple, time-efficient, and yields high exosome recovery and purity. Preferably, since the sample being processed is a solution system, a highly corrosion-resistant nickel mesh is chosen as the magnetic medium. After enriching the exosomes, the nickel mesh can be directly immersed in lysis buffer for in-situ lysis, and the supernatant is separated using a permanent magnet, achieving integrated exosome enrichment and nucleic acid extraction. Furthermore, considering the short and low abundance of target sequences in exosomal DNA, LNA-modified probes combined with digital PCR technology are used for detection. LNA modification significantly improves the binding affinity and single-base recognition specificity of the probe to the target sequence, while digital PCR enables absolute quantification of trace amounts of mutant DNA. Digital droplet PCR (ddPCR) is preferred for achieving ultra-high sensitivity detection of the target.

[0036] This invention addresses the clinical challenges in the subtyping and diagnosis of primary aldosteronism (PA), expanding the diagnostic scope from the generalized PA screening of existing technologies to a precise subtyping of APA subtypes. Since somatic mutations in the KCNJ5 gene (G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G)) are characteristic molecular events of APA subtypes and are rare in other subtypes such as IHA, this invention can directly distinguish APA from non-APA subtypes at the molecular level by detecting these mutation sites in plasma exosomes. Clinically validated, this method achieves a diagnostic specificity of 96.7% for APA subtypes and an area under the ROC curve (AUC) of 0.846, demonstrating high accuracy. The test results provide objective evidence for clinical treatment selection; a positive result suggests the patient may have an APA subtype that could benefit from surgery, while a negative result suggests a subtype such as IHA that requires drug treatment.

[0037] Compared to the current bilateral adrenal vein sampling (AVS) method for subtyping diagnosis, this invention only requires peripheral blood collection, completely avoiding the risks and complications associated with invasive procedures such as vascular puncture, contrast agent exposure, and adrenal vein rupture. Patients experience no pain, have high compliance, and there is no technical risk of catheter failure. This invention directly detects at the molecular level, significantly improving diagnostic accuracy and reliability. By integrating a high-gradient magnetic separation component, lysis reagent, and digital PCR detection reagent into a kit, the entire process from sample processing to result interpretation is standardized. The operation steps are clear, require minimal personnel skills, and have a short testing cycle, allowing for rapid implementation in medical institutions equipped with digital PCR instruments. This method can be combined with clinical indications, biochemical indicators, and imaging findings to provide a reliable basis for the comprehensive diagnosis and precise treatment planning of primary aldosteronism (PA), and is particularly suitable for large-scale screening and clinical promotion of APA subtypes in hypertensive individuals. Attached Figure Description

[0038] Figure 1 This is a flowchart illustrating the application of the diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection in Example 2 of the present invention.

[0039] Figure 2 ROC curves for diagnosing APA subtypes at three mutation sites in the KCNJ5 gene: G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G).

[0040] Figure 3 A statistical graph showing the sensitivity and specificity of detecting KCNJ5 gene mutations in clinical samples. Detailed Implementation

[0041] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0043] Example 1

[0044] This embodiment provides a diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection. The kit includes an exosome enrichment component, an exosome lysis component, a nucleic acid detection reagent, as well as calibrators and quality control products.

[0045] The exosome enrichment component includes enrichment reagents adapted to a high-gradient magnetic separation device. The high-gradient magnetic separation device includes a separation column with a flow cavity and a magnet disposed around the column to generate a magnetic field. A nickel mesh is placed within the flow cavity as a magnetic medium. Under the influence of the magnetic field, the nickel mesh generates a high-gradient magnetic field within the flow cavity to capture and enrich exosomes in the plasma sample. The enrichment conditions of the high-gradient magnetic separation device are: plasma sample volume 1 mL, plasma sample injected into the high-gradient magnetic separation device at a flow rate of 100 μL / min, magnetic separation time 10 min, and separation temperature 4 °C. Exosomes in the plasma are adsorbed and enriched on the nickel mesh under the influence of the magnetic field. After the sample passes through, the high-gradient magnetic separation component is disassembled, and the nickel mesh with exosomes attached is removed.

[0046] The exosome lysis reagent includes a lysis buffer for lysing exosomes to release nucleic acids. Transfer the nickel mesh to a centrifuge tube, then add 50 µL of PBS and 2 µL of a 20 mg / mL proteinase K solution, and incubate at 56 °C for 15 min to promote digestion. To inactivate proteinase K, heat the sample at 95 °C for 5 min using a PCR thermal cycler. A permanent magnet is set to adsorb the nickel mesh after lysis to separate the supernatant containing nucleic acids. The lysis buffer can be used immediately for ddPCR detection or stored at -80 °C.

[0047] The nucleic acid detection reagent includes LNA-modified specific probes, primer pairs, PCR reaction buffer, DNA polymerase, and dNTPs for detecting mutation sites in the KCNJ5 gene. Mutation sites include G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G).

[0048] The LNA-modified specific probes are designed for each mutation site as follows: For the G151R (c.451G>A) mutation site, the nucleotide sequence of the probe contains an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 1; for the G151R (c.451G>C) mutation site, the nucleotide sequence of the probe contains an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 2; and for the L168R (c.503T>G) mutation site, the nucleotide sequence of the probe contains an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 3.

[0049] Among them, SEQ ID NO: 1 is the target sequence of the KCNJ5 gene G151R (c.451G>A) mutant, SEQ ID NO: 2 is the target sequence of the KCNJ5 gene G151R (c.451G>C) mutant, and SEQ ID NO: 3 is the target sequence of the KCNJ5 gene L168R (c.503T>G) mutant, as detailed in Table 1. Information on KCNJ5 mutant primers and probes is detailed in Table 2.

[0050] The calibrators and quality control materials include standard plasmids containing specific mutation sites of the KCNJ5 gene as calibrators, and plasmids containing the wild-type KCNJ5 gene as quality control materials.

[0051] Table 1

[0052]

[0053] Table 2

[0054]

[0055] Example 2

[0056] like Figure 1 As shown, this embodiment provides a method for detecting a subtype of primary aldosteronism using the kit of Example 1, specifically including the following steps:

[0057] (1) Plasma sample preparation: Collect 2 mL of peripheral blood from the patient into an anticoagulant tube, place the anticoagulant tube in a centrifuge, centrifuge at 3000g for 15 min at 4℃, take the supernatant to obtain the plasma sample, and store the plasma sample at -80℃ for later use.

[0058] (2) Exosome enrichment and purification: Take 1 mL of plasma sample and inject it into the high gradient magnetic separation device in Example 1 at a flow rate of 100 μL / min. The magnetic separation time is 10 min and the separation temperature is 25 °C. Exosomes in the plasma are adsorbed and enriched on the nickel grid under the action of the magnetic field. After the sample has passed through, disassemble the high gradient magnetic separation device and take out the nickel grid with exosomes attached.

[0059] (3) Exosome lysis and nucleic acid extraction: Place the removed nickel mesh in a centrifuge tube, add lysis buffer, and ensure that the lysis buffer and nickel mesh are in full contact. The surfactant in the lysis buffer will disrupt the lipid bilayer vesicle structure of the exosomes, releasing the nucleic acid material inside the exosomes. After incubation, attach a permanent magnet to the bottom or side wall of the centrifuge tube to adsorb and fix the nickel mesh to the tube wall. Let it stand for a while to completely separate the nickel mesh from the liquid, and then extract the supernatant containing nucleic acid.

[0060] (4) Digital PCR detection: Prepare a digital PCR reaction system, namely 10 μL of 2× digital PCR premix, 1 μL of 20× primer-probe mixture (containing specific primers and LNA-modified probes for the three mutation sites G151R (c.451G>A), G151R (c.451G>C) and L168R (c.503T>G), and the nucleic acid extracted in step (3), and make up to 20 μL with nuclease-free water.

[0061] The reaction mixture was transferred to a digital droplet PCR chip and amplified using a digital droplet PCR instrument. The amplification program was set as follows: reverse transcription at 55°C for 20 min, pre-denaturation at 95°C for 2 min, denaturation at 95°C for 30 s, and annealing extension at 56°C for 30 s, for a total of 45 cycles.

[0062] After amplification, in situ multicolor fluorescence imaging was performed using a dPCR system at 58℃ to read fluorescence signals, analyze the fluorescence intensity of each reaction unit, and calculate the absolute copy number of the target mutant gene.

[0063] (5) Interpretation based on digital PCR test results: If one or more of the KCNJ5 gene mutation sites (G151R (c.451G>A), G151R (c.451G>C) and L168R (c.503T>G)) are positive (i.e. mutation signal is detected and copy number is higher than the preset threshold), it suggests that the subject may be a high-risk group for aldosterone-secreting adenoma (APA) subtype; if all test sites are negative, it suggests that the subject may be a non-APA subtype.

[0064] Verification Example

[0065] To verify the diagnostic efficacy of this invention based on plasma exosome KCNJ5 gene mutation detection for the aldosteronoma (APA) subtype of primary aldosteronism, an acquisition cohort and a validation cohort were established for validation.

[0066] 1. Obtain the queue:

[0067] Sample information: 105 patients with primary aldosteronism, all of whom had plasma and tumor tissue samples collected.

[0068] Detection Methods: Plasma samples were analyzed using the method described in Example 2 to detect three mutation sites (G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G)) in the KCNJ5 gene of exosomal samples. The three mutation sites of the KCNJ5 gene in tumor tissue samples were also detected. The accuracy of plasma exosome detection was evaluated based on the tissue analysis results.

[0069] Test results:

[0070] 1) Detection results of the G151R (c.451G>A) mutation site:

[0071] Of 105 patients with APA, tissue testing showed G151R (c.451G>A) mutation in 23 cases and negative in 82 cases. Plasma exosome testing showed that the mutation was detected in 16 of the 23 tissue-positive samples and not in 7; all 82 tissue-negative samples were negative. The detection accuracy for this site was 93.33% (98 / 105), the specificity was 100% (82 / 82), and the sensitivity was 69.57% (16 / 23). Figure 2 As shown, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.846 (95% CI: 0.762–0.909).

[0072] 2) Detection results of the G151R (c.451G>C) mutation site:

[0073] Tissue analysis revealed 12 positive cases and 93 negative cases of G151R (c.451G>C) mutation. Plasma exosome analysis showed that the mutation was detected in 7 of the 12 positive tissue samples and not in 5; all 93 negative tissue samples were negative. The detection accuracy for this site was 95.23% (100 / 105), the specificity was 100% (93 / 93), and the sensitivity was 58.33% (7 / 12). Figure 2 As shown, the AUC was 0.767 (95% CI: 0.674–0.844).

[0074] 3) Detection results of L168R (c.503T>G) mutation site:

[0075] Tissue analysis revealed 23 positive cases and 82 negative cases of the L168R (c.503T>G) mutation. Plasma exosome analysis showed that the mutation was detected in 16 of the 23 positive tissue samples and not in 7; in the 82 negative tissue samples, one was positive and the remaining 81 were negative. The detection accuracy for this site was 92.38% (97 / 105), the specificity was 98.78% (81 / 82), and the sensitivity was 69.57% (16 / 23). Figure 2As shown, the AUC was 0.802 (95% CI: 0.713–0.873).

[0076] Based on the combined analysis of the three mutation sites, a positive result at any one of the mutation sites is considered a positive result for the overall mutation. For example... Figure 3 As shown, the results indicate that the sensitivity of the detection of total KCNJ5 mutations in plasma exosomes was 63.83%, and the specificity was 96.7%.

[0077] The above results indicate that the detection results of KCNJ5 gene mutations in plasma exosomes are in good agreement with those in tissues. KCNJ5 mutations in exosomes can accurately reflect the gene mutation status and can serve as a reliable molecular marker for APA subtypes.

[0078] 2. Verification Queue

[0079] Sample Information: Plasma samples were collected from 120 patients with primary aldosteronism. All patients underwent bilateral adrenal vein sampling (AVS) for subtyping. Among them, 58 cases were diagnosed as APA subtype by AVS, and 62 cases were diagnosed as non-APA subtype (mainly IHA).

[0080] Detection Method: The method described in Example 2 was used to detect three mutation sites (G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G)) in the KCNJ5 gene of plasma exosomes. During the detection process, the operators were unaware of the patients' AVS genotyping results. After the detection was completed, each sample was pre-genotyped according to the interpretation criteria determined by the acquisition cohort (a positive result for any mutation site was considered a high risk for the APA subtype). The predicted subtype results from the plasma exosome detection were compared with the actual subtype determined by AVS to calculate the diagnostic efficacy index.

[0081] Results: Among 120 APA samples, plasma exosome KCNJ5 mutation detection correctly predicted 53 APA subtypes and 58 non-APA subtypes, with 4 false positives (mutation detected in non-APA patients) and 5 false negatives (mutation not detected in APA patients), for an accuracy rate of 92.50%.

[0082] The above results demonstrate that the method for detecting KCNJ5 gene mutations in plasma exosomes, as described in this invention, confirms a high degree of consistency between exosome mutations and tissue mutations in the acquisition cohort and exhibits excellent diagnostic efficacy (accuracy 92.50%) in the independent validation cohort. This method can accurately identify primary aldosteronism (PAA) subtypes, providing a reliable tool for non-invasive PA subtyping diagnosis and showing promising clinical application prospects.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of plasma exosome KCNJ5 gene mutation in the preparation of diagnostic reagents for subtypes of primary aldosteronism, characterized in that, The mutation sites of the KCNJ5 gene are selected from at least one of the following: G151R (c.451G>A), G151R (c.451G>C) and L168R (c.503T>G). The diagnostic reagent is used to detect the mutation status of the KCNJ5 gene in plasma exosomes. When one or more of the mutation sites are detected as positive, it indicates that the subject is a high-risk group for the primary aldosteronism subtype. The diagnostic reagent uses digital PCR technology to detect mutation sites in the KCNJ5 gene. The digital PCR technology uses LNA-modified specific probes. The reaction conditions for the digital PCR are: 55℃ reverse transcription for 20 min, 95℃ pre-denaturation for 2 min, 95℃ denaturation for 30 s, 56℃ annealing extension for 30 s, for a total of 45 cycles.

2. A diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection, characterized in that, The kit includes an exosome enrichment component, an exosome lysis reagent, and a nucleic acid detection reagent; The exosome enrichment component includes enrichment reagents adapted to a high-gradient magnetic separation device; the exosome lysis reagent is used to extract lysis buffers of nucleic acids released from exosomes; and the nucleic acid detection reagent is used to detect KCNJ5 gene mutation sites, the mutation sites including one or more of G151R (c.451G>A), G151R (c.451G>C), and L168R (c.503T>G). The enrichment conditions of the high gradient magnetic separation device are as follows: plasma sample volume is 1 mL, plasma sample is injected into the high gradient magnetic separation device at a flow rate of 50-200 μL / min, magnetic separation time is 5-20 min, and separation temperature is 25℃.

3. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 2, characterized in that, The nucleic acid detection reagent includes an LNA-modified specific probe for detecting KCNJ5 gene mutation sites, primer pairs, PCR reaction buffer, DNA polymerase, and dNTPs.

4. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 3, characterized in that, The LNA-modified specific probes are designed with sequences targeting each mutation site as follows: The probe targeting the G151R (c.451G>A) mutation site has a nucleotide sequence containing an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 1; The probe targeting the G151R (c.451G>C) mutation site has a nucleotide sequence containing an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 2; The probe targeting the L168R (c.503T>G) mutation site has a nucleotide sequence containing an LNA-modified fragment complementary to the sequence shown in SEQ ID NO: 3; Among them, SEQ ID NO: 1 is the target sequence of the KCNJ5 gene G151R (c.451G>A) mutant, SEQ ID NO: 2 is the target sequence of the KCNJ5 gene G151R (c.451G>C) mutant, and SEQ ID NO: 3 is the target sequence of the KCNJ5 gene L168R (c.503T>G) mutant.

5. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 2, characterized in that, The high gradient magnetic separation device includes a separation column with a flow cavity and a magnet disposed around the separation column to generate a magnetic field. A magnetic medium is provided inside the flow cavity. The magnetic medium can generate a high gradient magnetic field inside the flow cavity under the action of a magnetic field, so as to capture and enrich exosomes in the plasma sample.

6. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 5, characterized in that, The magnetic medium is one or more of nickel mesh, iron mesh, and cobalt mesh.

7. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 5, characterized in that, It also includes a permanent magnet, which is configured to adsorb and fix the magnetic medium after lysis to separate the supernatant containing nucleic acid.

8. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 2, characterized in that, The nucleic acid detection reagent is an amplification reagent suitable for digital PCR instruments, and the reaction conditions of the digital PCR are: reverse transcription at 55℃ for 20 min, pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 30 s, annealing and extension at 56℃ for 30 s, for a total of 45 cycles.

9. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 8, characterized in that, The digital PCR instrument is a digital droplet PCR instrument.

10. The diagnostic kit for primary aldosteronism subtypes based on plasma exosome KCNJ5 gene mutation detection as described in claim 2, characterized in that, The kit also includes calibrators and quality control samples. The calibrators are standard plasmids containing specific mutation sites of the KCNJ5 gene, and the quality control samples are plasmids containing the wild-type KCNJ5 gene.

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