massarray-based cardiovascular disease and antifungal medication guide test

Abstract

The application relates to the technical field of molecular detection technology, and particularly provides a detection product for cardiovascular diseases and anti-fungal drug guidance based on MassArray and application. The application can solve the problems of low throughput, high cost and incomplete information of existing single gene detection, and realizes one-time comprehensive evaluation of patient drug metabolism and pharmacodynamic gene background.

Description

Technical Field

[0001] This application relates to the field of molecular detection technology, and in particular to a nucleic acid mass spectrometry detection primer set, kit, and its application for guiding cardiovascular disease and antifungal drug use. Background Technology

[0002] Pharmacogenomics (PGx) aims to guide personalized medicine through genetic variation information and is a core component of precision medicine. Currently, over 100 genes have been identified as being associated with differences in response to more than 200 drugs, and the FDA has approved the inclusion of pharmacogenomics information in the package inserts of over 300 drugs.

[0003] In the field of cardiovascular disease treatment, individual differences in drug response are particularly significant. For example: ① 20%-30% of patients exhibit "resistance" to clopidogrel, leading to recurrent thrombotic events; ② The effective dose of warfarin varies by up to 20 times among individuals, and improper use can lead to bleeding or embolism; ③ The risk of statin-related myopathy is closely related to the SLCO1B1 genotype, with carriers of the C allele having a 4-10 times increased risk; ④ The efficacy and adverse reactions of ACE inhibitors vary significantly among patients with different ACE genotypes. Furthermore, antiepileptic drugs such as phenytoin, immunosuppressants such as tacrolimus, and antifungal drugs such as voriconazole are also highly dependent on CYP450 enzyme polymorphisms.

[0004] Currently, clinical methods for multi-site pharmacogenomics detection are mainly based on quantitative real-time PCR (qPCR) or sequencing. qPCR detects SNP sites by analyzing amplification curves using TaqMan probes or SYBR Green fluorescent dye. Although this method has been approved for many years and is relatively simple to operate, it has significant technical bottlenecks: it relies on changes in fluorescence signal intensity for genotyping, and the detection throughput is limited by the number of fluorescence channels in the instrument, typically detecting a maximum of 8 sites per run, and requiring different fluorescent probes for each target site; the interpretation of detection results depends on internal reference gene calibration, which is easily affected by differences in PCR amplification efficiency; furthermore, quality control depends on threshold settings such as Ct values, affecting the accuracy and stability of the results. Sanger genotyping, on the other hand, accurately genotypes SNPs by determining the base sequence of PCR products; although this technology is the "gold standard" for genotyping, its operation steps are cumbersome, the detection cycle is long, it cannot perform rapid detection of multiple sites simultaneously, and the reagent and equipment costs are high, making it unsuitable for rapid detection of large batches of clinical samples. While microarray hybridization / next-generation sequencing can achieve high-throughput, multi-site detection, it suffers from drawbacks such as high cost, complex operation, and long data analysis cycles, making it more suitable for research applications than routine clinical laboratory testing. Therefore, this application is submitted. Summary of the Invention

[0005] To address the aforementioned technical challenges, this application provides a primer set and reaction system for detecting corresponding polymorphic sites using a time-of-flight mass spectrometry platform, achieving compatibility and signal uniformity between multiplex PCR and single-base extension. It also provides a comprehensive interpretation scheme for detection results, outputting standardized drug use guidance based on the combined genotypes of 22 loci. This resolves the difficulty in balancing throughput, cycle time, and cost associated with existing detection methods.

[0006] The first objective of this application is to provide a specific primer set and its product for guiding detection of cardiovascular diseases and antifungal medication use.

[0007] The second objective of this application is to provide a method for guiding the detection of cardiovascular diseases and antifungal medication use.

[0008] To achieve the above objectives, this application provides the following technical solution: This application first provides a set of nucleic acid mass spectrometry primers for medication guidance, wherein the primer set consists of polymorphic sites of genes related to drug metabolism and efficacy in cardiovascular disease and antifungal treatment.

[0009] Furthermore, the polymorphic sites target 16 genes: CYP2C9, CYP2C19, CYP3A5, CYP4F2, SLCO1B1, PEAR1, ABCB1, VKORC1, ADD1, ADRB1, AGTR1, ACE, APOE, MTHFR, MTRR, and ALDH2.

[0010] Furthermore, the polymorphic sites include 22 sites: rs1799853, rs1057910, rs4986893, rs4244285, rs12248560, rs776746, rs2108622, rs4149056, rs12041331, rs1045642, rs9923231, rs4961, rs1801252, rs1801253, rs5186, I / D (287bp deletion), rs429358, rs7412, rs1801131, rs1801133, rs1801394, and rs671.

[0011] In some respects, the primer set includes amplification primers, the sequences of which are shown in SEQ ID NO. 1-45, or have at least 85% identity with SEQ ID NO. 1-45.

[0012] Furthermore, the primer set also includes extension primers, the sequences of which are shown in SEQ ID NO.46-65, or have at least 85% identity with SEQ ID NO.46-65.

[0013] This application also provides a product comprising the primer set described in any of the preceding claims.

[0014] In some respects, the product is in the form of a reagent kit.

[0015] In some respects, the product also includes reagents and / or equipment for detecting indicators; More preferably, the reagents include: 10×PCR Buffer, dNTP Mix, MgCl2, Primer Mix, PCR Enzyme, and nuclease-free water; Furthermore, the device is a MassARRAY nucleic acid mass spectrometer.

[0016] This application also provides the application of the aforementioned primer set in nucleic acid mass spectrometry detection for cardiovascular and antifungal drug guidance.

[0017] This application also provides the application of the aforementioned primer set in the preparation of nucleic acid mass spectrometry detection kits for cardiovascular and antifungal drug use guidance.

[0018] This application also provides a method for guiding detection of cardiovascular and antifungal drugs. The specific method includes: using the above-mentioned primer set to perform PCR amplification and base extension reaction on the genome of the sample to be tested, and then using MassARRAY to detect the product obtained from the reaction to determine the genotype of the drug resistance gene mutation site in the sample to be tested.

[0019] Compared with the prior art, this application has the following technical advantages.

[0020] 1) This application has scientifically selected and combined the types and specific loci of genes that guide cardiovascular diseases and antifungal drug use through exploratory research, achieving coverage of 16 genes and 22 loci in a single test. It is one of the mass spectrometry reagent kits with the widest coverage and the most clinical application scenarios among the cardiovascular and antifungal pharmacogenomics testing products in China. It truly realizes "one test, lifelong guidance for multiple drugs". Patients can obtain lifelong medication guidance information for multiple drug-related genes by performing this test once in their lifetime, avoiding repeated blood collection and repeated testing, saving medical resources and patient costs.

[0021] 2) This application designs 22 pairs of amplification primers and 22 single-base extension primers for 22 target sites. The primer design of this application fully considers the cross-matching between primers in the multiplex PCR system, the uniformity of annealing temperature, the absence of primer dimer formation, and the molecular weight difference of the extension products being greater than 30 Da. In terms of specific screening and optimization, this application has compared and optimized each set of primer sequences to ultimately ensure the signal-to-noise ratio and interpretation accuracy of the mass spectrometry peaks, and truly realize the simultaneous specific and effective detection of the test samples using the MassARRAY system.

[0022] 3) This application boasts high throughput and low cost. Based on time-of-flight mass spectrometry technology, it can complete the testing of 384 samples in a single 384-well plate test, with reports issued within 7 hours. The reagent cost per sample is controlled at 1 / 5 to 1 / 3 of that of traditional qPCR single-gene testing, demonstrating significant health economic benefits. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 Results before and after primer testing targeting the rs4986893 site.

[0025] Figure 2 Results before and after primer testing targeting the rs4244285 site.

[0026] Figure 3 The results of nucleic acid mass spectrometry detection of samples P1-P5.

[0027] Figure 4 The results of first-generation sequencing of samples P1-P5. Detailed Implementation

[0028] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Furthermore, the embodiments described are only some, not all, of the embodiments of this application. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] In this application, the MassARRAY gene analysis technology is based on MALDT-TOF time-of-flight mass spectrometry. First, the target gene is enriched by PCR amplification. Then, the extension primer extends the target fragment by one base. Finally, the genotype of the detected site can be obtained by comparing the difference in the quality of the single-base extension product.

[0030] The present application is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present application in any way.

[0031] To establish a multiplex nucleic acid mass spectrometry detection system for personalized drug use guidance for cardiovascular diseases and antifungal diseases, the applicant first conducted a systematic target site screening. The overall process was mainly divided into two stages.

[0032] Phase 1: Literature systematic review and database mining to establish initial screening inclusion criteria and initial screening candidate loci.

[0033] The applicant established the following initial screening inclusion criteria by searching databases such as PubMed, PharmGKB, CPIC (Clinical Pharmacogenetics Implementation Consortium), and DPWG (Dutch Working Group on Pharmacogenetics): 1) Drugs that are clearly related to the metabolism, efficacy, and toxicity of cardiovascular drugs (antithrombotic, antihypertensive, and lipid-lowering drugs) and include other indications targeting these drugs, such as azole antifungals; 2) Supported by at least one clinical association study or meta-analysis with ≥100 cases; 3) Minor allele frequency (MAF) ≥1% in East Asian populations.

[0034] Based on this, a total of 86 candidate sites (involving 38 genes) were initially obtained, and some representative preliminary screening results are shown in Table 1 below.

[0035] Table 1. Information on some representative initial screening sites

[0036] Phase Two: Multidimensional weighted scoring, including allele frequency, level of clinical evidence, functional importance, and multiple compatibility assessment.

[0037] In order to select the core loci most suitable for the multiplex detection system from the initial screening loci, the applicant established an internal quantitative scoring system, which scores each locus from five dimensions in Table 2 below (1-5 points for each item, total 25 points).

[0038] Table 2. Quantitative Scoring System

[0039] Specific screening thresholds: Loci with a total score ≥18 points and A ≥3 points, B ≥3 points, and E ≥3 points are included in the final system; for some functionally important but slightly low-frequency loci (such as CYP2C19 rs12248560, although the frequency is only 5%, the evidence is strong and the intervention value is high), they are included after weighing the pros and cons.

[0040] Ultimately, this application identified 22 sites with an average score of 20.5 and a high recommendation level (1A or 1B). Specific target site information is shown in Table 3 below.

[0041] Table 3. Information on the Specific Target Sites Identified

[0042] The 22 target sites cover a total of 16 genes, involving the following drug / disease areas, as shown in Table 4 below.

[0043] Table 4. Drug types corresponding to the 22 target sites

[0044] The integrated design of the above sites enables this primer system to simultaneously guide the individualized use of six major classes of cardiovascular-related drugs, including antithrombotic, antihypertensive, lipid-lowering, and antifungal drugs, truly realizing the clinical value of "one sampling, one test, and multi-drug guidance".

[0045] Example 2: Design and screening of the primer sequence system of this application For the identified target sites, this application initially designed a batch of MassARRAY amplification and extension primer sequences based on this target site information, followed by performance evaluation and sequence optimization. Due to space limitations, only the optimization and screening of rs4986893 and rs4244285 sites are used as examples.

[0046] 1) Primer for rs4986893 site Amplification primers designed for the rs4986893 locus were tested, and the results of detection of heterozygous samples at the rs4986893 locus were as follows (see [link to test]). Figure 1 The initial primer combination 1 showed inconsistent performance in extending the G and A products, exhibiting high and low peaks, resulting in unsatisfactory results. However, the adjusted primer sequence combination 2 achieved basically consistent performance in extending the G and A products, yielding more ideal results. The two primer sequences are shown in the table below.

[0047]

[0048] 2) Primer at rs4244285 site For the rs4244285 site, tests showed that primer combination 1 before optimization exhibited inconsistent performance in the extension of G and A products, showing a high-low peak phenomenon, with a high G peak and a low A peak, or vice versa (see [link to product details]). Figure 2 The primers were subsequently optimized, and the performance of the extended G and A products of primer combination 2 was basically consistent, which was quite ideal. Therefore, primer combination 2 was included in the system construction. The primer sequences before and after optimization are shown in the table below.

[0049]

[0050] Through the aforementioned series of primer design and optimization experiments, this application finally established the amplification primer system and the extension primer system (see Table 5).

[0051] Table 5. List of Amplification Primer Sequences

[0052] The extended primer information is shown in Table 6 below.

[0053] Table 6. List of Extended Primer Sequences

[0054] Based on the determined amplification primers and extension sequences, the applicant subsequently synthesized primer compounds and stored them for future use.

[0055] Example 3: Establishment of the detection kit and determination of the detection method of this application This application utilizes the primers finally synthesized in Example 2, along with standard MassARRAY components such as 10×PCR Buffer, dNTP Mix, MgCl2, Primer Mix, PCR Enzyme, and nuclease-free water, to prepare a convenient kit for experimental detection. A corresponding nucleic acid mass spectrometry detection procedure guided by cardiovascular / antifungal medication guidelines has also been established. The detailed detection procedure is as follows: 1) Sample processing and DNA extraction 2-4 mL of peripheral venous blood was collected from the subject (collected in EDTA anticoagulant tubes), and genomic DNA was extracted using a commercially available genomic DNA extraction kit (such as the Qiagen QIAamp DNA Mini Kit or an equivalent method). The extracted DNA was then analyzed by spectrophotometry (OD260 / 280 between 1.7 and 2.0) and quality controlled by agarose gel electrophoresis, before being diluted to a concentration of 5-20 ng / μL.

[0056] 2) DNA amplification and extension After quality control, the DNA samples were subjected to multiplex PCR amplification, and the reaction system is shown in the table.

[0057]

[0058] The PCR amplification procedure is shown in the table below.

[0059]

[0060] Enzymatic hydrolysis was then carried out, and the enzymatic hydrolysis reaction system is shown in the table below.

[0061]

[0062] The enzymatic hydrolysis reaction conditions are shown in the table below.

[0063]

[0064] Finally, an extension reaction is carried out, and the extension reaction system is shown in the table below.

[0065]

[0066] The conditions for the extended reaction are shown in the table below.

[0067]

[0068] 3) Mass spectrometry detection and analysis: The purified product is spotted onto the detection chip and detected by mass spectrometry in flight. After detection, the data is analyzed using the accompanying software to obtain the results.

[0069] Example 4: Performance Verification of the Detection System This embodiment uses the method and procedure of Example 3 to verify the performance (including accuracy and precision) of the reagent kit.

[0070] 1. Accuracy Regarding the accuracy evaluation, the applicant tested 14 clinical samples with a concentration of 20 ng / μL at each point and compared the mass spectrometry results with the sequencing results.

[0071] For the test results of some samples (P1-P5), please refer to [link / reference needed]. Figure 3 and Figure 4 The results are shown in Table 7 below. It can be seen that the mass spectrometry detection results of 22 SNP sites in 14 clinical samples are completely identical to the first-generation sequencing detection results, with a consistency of 100%, which is in line with expectations.

[0072] Table 7. Mass spectrometry and first-generation sequencing results for samples P1-P6.

[0073] 2. Precision For precision evaluation, the applicant used three batches of reagents to repeatedly test four samples, with three repetitions, to verify intra-batch and inter-batch precision. During the evaluation process, the four samples were tested three times within the same batch to compare intra-batch precision; the same operator tested the four identical samples simultaneously in three batches to compare inter-batch precision.

[0074] The evaluation results showed that the intra-batch and inter-batch mass spectrometry results of the four clinical samples were 100% consistent, and the precision met the expected target (see Table 8).

[0075] Table 8. Intra-batch precision results

[0076] The results of batch-to-batch precision are shown in Table 9.

[0077] Table 9. Inter-batch precision results

[0078] 1. Clinical validation 1 - Targeting clopidogrel The applicant completed clinical trials at four institutions: the First Affiliated Hospital of Zhengzhou University, the First Hospital of Nanjing, the First Hospital of Peking University, and the First Affiliated Hospital of Xinjiang Medical University. The clinical performance of the product was confirmed by comparing the reagent kit of this application with a commercially available fluorescent probe method product (Human CYP2C19 Gene Polymorphism Detection Kit (PCR-Fluorescent Probe Method), National Medical Device Registration Certificate No. 20173403155).

[0079] The enrolled sample consisted of 1757 patients with coronary artery disease or those currently taking clopidogrel. For the CYP2C19*2 locus (rs4244285, c.681G>A), there were 893 cases of the GG genotype, 716 cases of the GA genotype, and 148 cases of the AA genotype; for the CYP2C19*3 locus (rs4986893, c.636G>A), there were 1578 cases of the GG genotype, 172 cases of the GA genotype, and 7 cases of the AA genotype; for the CYP2C19*17 locus (rs12248560, c.-806C>T), there were 1673 cases of the CC genotype, 80 cases of the CT genotype, and 4 cases of the TT genotype.

[0080] The statistical results of each genotype test are shown in Table 10 below.

[0081] Table 10. Detection results of different gene loci based on this kit and the control kit.

[0082] The test results showed that, for the detection of polymorphisms at the aforementioned three gene loci, the concordance rate between this product and the control reagent was 100% for each genotype, with an overall concordance rate of 100%. Clinical trial results showed that this product had good consistency with similar products already on the market.

[0083] 2. Clinical validation - for use with voriconazole The applicant also completed a clinical trial at the First Affiliated Hospital of Xinjiang Medical University, comparing the reagent kit of this application with the clinical reference method (Sanger sequencing) to confirm the clinical performance of the product.

[0084] The enrolled samples were patients currently taking or about to take voriconazole for antifungal treatment, totaling 63 patients. For the CYP2C19*2 locus (rs4244285, c.681G>A), there were 32 cases of GG genotype, 26 cases of GA genotype, and 5 cases of AA genotype; for the CYP2C19*3 locus (rs4986893, c.636G>A), there were 56 cases of GG genotype, 7 cases of GA genotype, and 0 cases of AA genotype; for the CYP2C19*17 locus (rs12248560, c.-806C>T), there were 56 cases of CC genotype, 7 cases of CT genotype, and 0 cases of TT genotype. The statistical results of each genotype are shown in Table 11 below.

[0085] Table 11. Detection results of different gene loci based on this kit and Sanger sequencing.

[0086] The test results showed that, for the detection of polymorphisms at the aforementioned three gene loci, the concordance rate between this product and the control reagent was 100% for each genotype, with an overall concordance rate of 100%. Clinical trial results showed that this product was in good agreement with the clinical reference method.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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 therein. Such 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 this application.

Claims

1. A primer set for nucleic acid mass spectrometry detection for medication guidance, characterized in that, The primer set consists of polymorphic sites of genes related to drug metabolism and efficacy in cardiovascular disease and antifungal treatment.

2. The primer set according to claim 1, characterized in that, The polymorphic sites target 16 genes: CYP2C9, CYP2C19, CYP3A5, CYP4F2, SLCO1B1, PEAR1, ABCB1, VKORC1, ADD1, ADRB1, AGTR1, ACE, APOE, MTHFR, MTRR, and ALDH2; the polymorphic sites target 22 sites: rs1799853, rs1057910, rs4986893, rs4244285, rs12248560, rs776746, rs2108622, rs4149056, rs12041331, rs1045642, rs9923231, rs4961, rs1801252, rs1801253, rs5186, I / D (287bp) (deletion), rs429358, rs7412, rs1801131, rs1801133, rs1801394 and rs671.

3. The primer set according to claim 2, characterized in that, The primer set includes amplification primers, the sequences of which are shown in SEQ ID NO.1-45, or have at least 85% identity with SEQ ID NO.1-45.

4. The primer set according to claim 2, characterized in that, The primer set further includes extension primers, the sequences of which are shown in SEQ ID NO.46-65, or have at least 85% identity with SEQ ID NO.46-65.

5. A product characterized in that, Includes the primer set as described in any one of claims 1-4.

6. The product according to claim 5, characterized in that, The product is in the form of a reagent kit.

7. The product according to claim 5, characterized in that, The product also includes reagents and / or equipment for detecting indicators; preferably, the reagents include: 10×PCR Buffer, dNTP Mix, MgCl2, Primer Mix, PCREnzyme, and nuclease-free water; the equipment is a MassARRAY nucleic acid mass spectrometer.

8. The application of the primer set according to any one of claims 1-4 in nucleic acid mass spectrometry detection guided by cardiovascular and antifungal medications.

9. The use of the primer set according to any one of claims 1-4 in the preparation of a nucleic acid mass spectrometry detection kit for cardiovascular and antifungal drug use guidance.

10. A method for guiding the detection of cardiovascular and antifungal medication use, characterized in that, The method includes using the primer sets described in any one of claims 1-4 to perform PCR amplification and base extension reaction on the genome of the sample to be tested, and then using MassARRAY to detect the product obtained from the reaction to determine the genotype of the drug resistance gene mutation site in the sample to be tested.

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