Kit and method for bromhidrosis or / and earwax type related ABCC11 gene polymorphism detection

Abstract

The invention discloses a kit and a method for detecting bromhidrosis or / and earwax type related ABCC11 gene polymorphism. The kit comprises two pairs of primers for detecting bromhidrosis or / and earwax type related ABCC11 gene mutation and corresponding probes. The kit is based on an amplification block mutation system real-time fluorescent quantitative PCR (ARMS-qPCR) technology, the bromhidrosis or / and earwax type related ABCC11 gene mutation type can be accurately and rapidly detected, the bromhidrosis or / and earwax type gene detection efficiency is improved, and the operation method adopting the kit for detection is simple, rapid and suitable for large-scale detection; when the kit is used for detection, the misjudgment rate is reduced, subjective errors are avoided, and immediate treatment or prevention measures are provided for a patient with bromhidrosis.

Description

Technical Field

[0001] This invention belongs to the field of in vitro nucleic acid detection technology, and relates to a gene polymorphism detection kit and detection method, specifically a kit and detection method for detecting ABCC11 gene polymorphism related to axillary odor and / or earwax. Technical Background

[0002] Axillary osmidrosis (AO) is a common body odor disorder characterized by a pungent odor produced when secretions from the apocrine sweat glands in the armpits are broken down by bacteria on the skin surface. It seriously affects the mental health and social activities of patients.

[0003] Recent studies have found a close association between axillary osmidrosis (body odor) and the ABCC11 gene polymorphism rs17822931. The single nucleotide variant c.538G>A at this site directly affects the secretory function of apocrine sweat glands. Individuals carrying the G allele (GG or GA type) typically exhibit axillary osmidrosis, while those carrying the AA allele do not. Furthermore, this genotype is also associated with wet earwax, while individuals carrying the AA allele typically exhibit dry earwax.

[0004] Traditional methods for diagnosing bromhidrosis (body odor) rely primarily on subjective evaluations of the patient's body odor and clinical examinations, lacking clear laboratory diagnostic criteria and potentially leading to misdiagnosis. Genetic testing, on the other hand, provides objective diagnostic evidence, avoids subjective errors, and identifies high-risk individuals before symptoms appear, allowing for early preventative measures. Furthermore, genetic testing provides a basis for genetic counseling for families with a history of bromhidrosis, helping individuals better plan their reproductive decisions and providing scientific genetic guidance for future generations.

[0005] Currently, gene testing for axillary odor mainly employs Sanger sequencing or a combination of high-throughput sequencing and Sanger sequencing. However, these methods suffer from cumbersome procedures, high testing costs, and limited accessibility. Therefore, rapid, simple, and cost-effective quantitative real-time PCR (qPCR) has become the primary method in the field of molecular diagnostics.

[0006] In recent years, qPCR combined with loop-mediated isothermal amplification (LAMP) technology has been reported for use in the detection of axillary odor. LAMP technology is suitable for rapid on-site detection due to its high amplification efficiency, low equipment requirements, and ease of operation. However, the high sensitivity of LAMP technology also makes it extremely sensitive to aerosol contamination, easily leading to false positive results, which is a major challenge in its practical application. In addition, methods using melting curve analysis have been reported in China, but this method requires DNA extraction, thus affecting the detection time.

[0007] To address these issues, we propose a method for detecting axillary osmidrosis and / or earwax-related genes based on Amplification Refractory Mutation System qPCR (ARMS-qPCR) technology. The core of this method lies in designing specific primers whose 3' ends are completely complementary to the target mutation site. If the bases at the 3' end of the primer match the template, the target template can be amplified; otherwise, it cannot. This technology has proven reliable and has been widely applied. This method requires a small sample size and is fast, requiring only a drop of blood or an oral swab sample from the patient, providing results within one hour. It is simple to operate and highly accurate. The development of this rapid detection method is of great significance for the early diagnosis of axillary osmidrosis, genetic counseling, and the formulation of personalized treatment plans. Summary of the Invention

[0008] The purpose of this invention is to address the problems of cumbersome procedures, complex result analysis, high testing costs, and poor accessibility in existing detection methods for ABCC11 gene mutation polymorphisms in axillary odor and / or earwax formation. This invention provides a detection kit and method based on amplification arrest mutation system qPCR technology for detecting ABCC11 gene mutation polymorphisms in axillary odor and / or earwax formation. The detection kit of this invention is rapid, simple, and cost-effective for detecting ABCC11 gene mutations in axillary odor and / or earwax formation, making it suitable for large-scale testing. Furthermore, the kit of this invention exhibits high specificity and sensitivity in detecting ABCC11 gene mutations in axillary odor and / or earwax formation.

[0009] One aspect of this invention provides a kit for detecting gene polymorphisms associated with axillary odor and / or earwax, comprising two pairs of primers and corresponding probes for detecting ABCC11 gene mutations associated with axillary odor and / or earwax, referred to as primer-probe combination pairs.

[0010] The mutation site of the ABCC11 gene associated with axillary odor and / or earwax is rs17822931.

[0011] In particular, the two primer-probe pair used to detect ABCC11 gene mutations associated with axillary odor and / or earwax are c.538G and c.538A.

[0012] In particular, the forward primer sequence of the c.538G primer-probe combination is SEQ ID NO.1; the reverse primer sequence is SEQ ID NO.2; and the probe sequence is SEQ ID NO.3.

[0013] In particular, the forward primer sequence of the c.538A primer-probe combination is SEQ ID NO.1; the reverse primer sequence is SEQ ID NO.4; and the probe sequence is SEQ ID NO.3.

[0014] In particular, the nucleotide sequence of the forward primer in the c.538G primer-probe combination is: GCTTCACCGCCTTTGGGAA; the nucleotide sequence of the reverse primer is: TCTGCCACTTAC TGGCGC; and the nucleotide sequence of the probe is: 5`6-FAM-AGTACACTGGCAATGCAGAAG CAGA TG-3`BHQ1.

[0015] In particular, the nucleotide sequence of the forward primer in the c.538A primer-probe combination is: GCTTCACCGCCTTTGGGAA; the nucleotide sequence of the reverse primer is: AGTCTGCCACT TACTGGCGT; and the nucleotide sequence of the probe is: 5`6-FAM-AGTACACTGGCAATGCAG AAGCAGATG-3`BHQ1.

[0016] In particular, the primers are qPCR primers designed based on the mutation sites of the detected genes; the probes are TaqMan probes designed based on the mutation sites of the detected genes.

[0017] In particular, the kit also includes a positive control, wherein the positive control is DNA extracted from human embryonic kidney cells 293T, and Sanger sequencing has confirmed that the rs17822931 site in the ABCC11 gene is of the GG genotype.

[0018] In particular, the kit also includes a negative control, wherein the negative control is DNA extracted from human gallbladder cancer cells GBC-SD, and Sanger sequencing has confirmed that the rs17822931 site in the ABCC11 gene is of the AA genotype.

[0019] In particular, the kit also includes qPCR premix.

[0020] In particular, the qPCR premix is ​​preferably Novizan qPCR premix, 2 × Taq Pro U + MultipleProbe qPCR Mix, catalog number: QN213.

[0021] Another aspect of the present invention provides the application of the above-described kit in the detection of mutations in the ABCC11 gene of axillary odor and / or earwax.

[0022] Another aspect of the present invention provides a method for detecting the ABCC11 gene mutation polymorphism of axillary odor and / or earwax using the above-mentioned kit, comprising the following steps:

[0023] 1) Release total DNA from anticoagulated whole blood or oral swabs to obtain sample DNA;

[0024] 2) Place the reagents from the kit and the sample DNA into a qPCR reaction tube, perform a qPCR reaction, and interpret the gene mutation site based on the measured CT value to determine the type of ABCC11 gene mutation.

[0025] In particular, in step 1), DNA extraction kits are used to extract DNA from anticoagulated whole blood or oral swabs.

[0026] In particular, while performing the qPCR reaction in step 2), the positive control and negative control are mixed with the reagents in the kit, placed in a qPCR reaction tube, and the qPCR reaction is performed and the fluorescence is detected.

[0027] In particular, if the CT value of the qPCR reaction using the c.538G primer-probe combination in the kit is ≤35 in step 2), and the CT value of the qPCR reaction using the c.538A primer-probe combination in the kit is >35, it indicates that the genotype of the sample being tested is GG, and the result is positive.

[0028] In particular, if the CT value of the qPCR reaction using the c.538G primer-probe combination in the kit is ≤35 in step 2), and the CT value of the qPCR reaction using the c.538A primer-probe combination in the kit is ≤35, then the genotype of the sample being tested is GA, and the result is positive.

[0029] In particular, if the CT value of the qPCR reaction using the c.538G primer-probe combination in the kit in step 2) is >35, and the CT value of the qPCR reaction using the c.538A primer-probe combination in the kit is ≤35, then the genotype of the sample being tested is AA and is negative.

[0030] In particular, during the qPCR reaction in step 2), the reaction system is as follows:

[0031] In particular, the qPCR reaction procedure is as follows: contamination digestion, 37℃, 2 min; pre-denaturation: 95℃, 30 s; denaturation-extension cycle reaction, 45 cycles: ① denaturation: 95℃, 10 s; ② extension: 60℃, 30 s.

[0032] The ABCC11 gene mutation polymorphism detection kit and detection method related to axillary odor and / or earwax of the present invention have the following advantages and benefits: 1. The ABCC11 gene mutation polymorphism detection kit for axillary odor and / or earwax type of the present invention provides a new idea and direction for the detection of axillary odor and / or earwax type.

[0033] 2. The gene detection site of the kit for detecting ABCC11 gene mutation polymorphism of axillary odor and / or earwax type of the present invention is reasonably selected and is a recognized mutation site related to axillary odor and / or earwax type, and has universality.

[0034] 3. The kit of this invention is used to detect genes related to axillary odor and / or earwax, avoiding subjective misjudgment of axillary odor and enabling timely treatment or prevention measures.

[0035] 4. Gene detection using the kit of this invention is simple, accurate, low-cost, widely applicable, and highly popular.

[0036] 5. The kit of this invention can be used for multiple gene detections, is less prone to contamination, and provides reliable results, making it suitable for long-term, large-sample detection. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0038] Example 1: Design of gene mutation primers

[0039] This invention targets and genotypes the rs17822931 locus of the ABCC11 gene associated with axillary osmidrosis and / or earwax. Two sets of primer and probe combinations were designed: one set to detect the G allele at the rs17822931 locus (i.e., the presence of the G type), and the other set to detect the A allele (i.e., the presence of the A type).

[0040] The sequence of the ABCC11 gene was obtained from NG_011522.2 in the NCBI database.

[0041] Primers designed by placing the 3' end of the primer at the detection site and artificially altering the penultimate base will amplify non-specific products with the reverse primer formed by the primer itself. These products cannot be detected using the Sybrgreen method and require the introduction of Taqman probes to obtain specific amplification products.

[0042] Primers were designed using the above methods, and the primer and probe combination with the highest discrimination was selected from them (i.e., the two primer and probe combinations with the highest discrimination for the A allele and the G allele), namely c.538G and c.538A.

[0043] The nucleic acid sequences of the primers and probes used in this invention for analyzing and detecting ABCC11 gene mutations are shown in Table 1.

[0044] Table 1 Primer and probe combinations

[0045] Wherein: FAM is the fluorescent group, BHQ1 is the quenching group; in addition to FAM, the fluorescent group at the 5' end of the probe can also be VIC, ROX, Cy5 or HEX; the quenching group at the 3' end can also be BHQ2, BHQ3 or TAMRA, and the excitation wavelength of the fluorescent group is matched with the absorption wavelength of the quenching group.

[0046] Example 2: Construction of negative and positive quality control samples

[0047] The negative control was DNA extracted from GBC-SD (human gallbladder cancer cells) (Pronosai, catalog number: CL-0085), and Sanger sequencing confirmed that the rs17822931 site in its ABCC11 gene was of the AA genotype.

[0048] The positive control was DNA extracted from 293T (human embryonic kidney cells) (Pronosai, catalog number: CL-0005), and Sanger sequencing confirmed that the rs17822931 site in its ABCC11 gene was of the GG genotype.

[0049] This invention uses the QIAamp® Blood DNA Extraction Kit to extract the corresponding DNA according to the kit's operating instructions.

[0050] Example 3

[0051] 1. Sample processing

[0052] Use the Room Temp Sample Lysis Kit (Novozymes, catalog number: P073) to process EDTA anticoagulant whole blood or oral swabs. Sample processing methods and procedures must strictly follow the kit's operating instructions.

[0053] EDTA anticoagulant in whole blood: 1) Take 2 μl of EDTA anticoagulant whole blood into a centrifuge tube, add 20 μl of Lysis Buffer (Novozymes, catalog number: P073), and mix by inverting. 2) Incubate at room temperature (20 ~ 25°C) for 3 min; 3) Add 20 μl of Stabilizing Buffer (Novozymes, catalog number: P073), invert and mix well to obtain the lysed DNA solution, and prepare the processed sample.

[0054] Oral swab: 1) Place the oral swab in a centrifuge tube containing 400 μl of Lysis Buffer (Novozymes, catalog number: P073) and mix by inverting. 2) Incubate at 95°C for 3 min; 3) Add 400 μl of Stabilizing Buffer (Novozymes, catalog number: P073), invert and mix well to obtain the lysed DNA solution and prepare the processed sample.

[0055] 2. qPCR process

[0056] 2-1 Sample Configuration

[0057] The processed samples, the positive control and negative control prepared in Example 2 were placed in qPCR tubes and mixed as shown in Table 2. Table 2 qPCR reaction system

[0058] The processed samples / positive controls / negative controls should be tested simultaneously in different reaction wells to ensure the reliability of the test results. The final concentration of the positive and negative controls should not be too high; a final concentration of 0.75 ng / μl is recommended (usually 1-0.1 ng / μl).

[0059] 2-2. qPCR reaction, where the qPCR procedure is shown in Table 3; Table 3 qPCR reaction procedure

[0060] 2-3. Interpretation of qPCR results

[0061] First: Interpretation of quality control results to ensure the reliability of test sample results. The interpretation criteria are as follows: A: For primer c.538G group, the positive control product Ct value is ≤35, and the negative reference product CT value is >35; B: For primer c.538A group, the negative control Ct value is >35, and the negative reference CT value is ≤35.

[0062] If the results do not match (e.g., contamination exists), the experimental results are invalid, and it is recommended to repeat the experiment.

[0063] If the results meet this requirement, the sample results can be interpreted.

[0064] Secondly: Interpretation of sample results: a: The Ct value of the sample detected by primer c.538G group was ≤35, and the Ct value of the sample detected by primer c.538A group was >35, and the genotype of the sample was GG.

[0065] b: The Ct value of the sample detected by primer c.538G group was ≤35, the Ct value of the sample detected by primer c.538A group was ≤35, and the genotype of the sample was GA.

[0066] c: The Ct value of the sample detected by primer c.538G group was >35, and the Ct value of the sample detected by primer c.538A group was ≤35, and the genotype of the sample was AA.

[0067] Experimental Example 1: Sample Detection

[0068] The actual samples were tested using a qPCR instrument to determine the genotype of the samples, and the results were compared with those from Sanger sequencing. The specific procedure is as follows: 1. Sample DNA extraction DNA was released from anticoagulated whole blood or oral swabs of 6 suspected axillary osmidrosis patients at the Institute of Dermatology, Chinese Academy of Medical Sciences using the Room Temp Sample Lysis Kit (Novozymes, catalog number: P073). A total of 6 samples were obtained and labeled as YB1-6. 2. Take out one 96-well plate or two 8-tube qPCR tubes, add reagents to each well as shown in Table 2, and briefly centrifuge and mix. 3. Place the 96-well plate or two 8-tube qPCR tubes into the qPCR instrument (QuantStudio™ 5 Real-Time Quantitative PCR System, 96-well, 0.1 mL, catalog number: A28573), set the reaction program as shown in Table 3, perform the qPCR reaction, and measure the CT value of each sample; 4. Based on the measured CT values, the genotyping of samples 1-6 and the control samples was analyzed, and the results are shown in Table 4.

[0069] Table 4. qPCR test results of the samples

[0070] The detection results of the kit of this invention are the same as those obtained using Sanger sequencing. This indicates that the kit of this invention has high accuracy. Specific results are shown in Table 5.

[0071] Table 5 Comparison of Sample Genotypes and Sanger Sequencing Results

[0072] Samples 1-6 were sequenced using the Sanger sequencing method, and the detection results of the kit of this invention were the same as those obtained using Sanger sequencing. This indicates that the kit of this invention has high accuracy and specificity.

[0073] The above embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A kit for detecting gene polymorphisms in axillary odor and / or earwax type, characterized in that, It includes two pairs of primers and corresponding probes for detecting ABCC11 gene mutations that cause bromhidrosis and / or earwax, referred to as primer-probe pair.

2. The kit according to claim 1, characterized in that, The two primer-probe pairs used to detect ABCC11 gene mutations that cause bromhidrosis and / or earwax are c.538G and c.538A. The forward primer sequence of the c.538G primer-probe pair is SEQ ID NO.1; the reverse primer sequence is SEQ ID NO.2; and the probe sequence is SEQ ID NO.

3. The forward primer sequence of the c.538A primer-probe pair is SEQ ID NO.1; the reverse primer sequence is SEQ ID NO.4; and the probe sequence is SEQ ID NO.

3.

3. The kit as described in claim 1 or 2, characterized in that, It also includes positive control samples, wherein the positive control samples are DNA extracted from human embryonic kidney cells 293T, and Sanger sequencing has confirmed that the rs17822931 site in the ABCC11 gene is of the GG genotype.

4. The kit as described in claim 1 or 2, characterized in that, It also includes negative control samples, wherein the negative control samples are DNA extracted from human gallbladder cancer cells GBC-SD, and Sanger sequencing has confirmed that the rs17822931 site in the ABCC11 gene is of the AA genotype.

5. The application of a kit as described in any one of claims 1-4 in the detection of ABCC11 mutations in the axillary odor and / or earwax type gene.

6. A method for detecting ABCC11 gene mutation polymorphism of axillary odor and / or earwax type using the kit as described in claim 1, characterized in that, Includes the following steps: 1) Release total DNA from anticoagulated whole blood or oral swabs to obtain sample DNA; 2) Place the reagents from the kit and the sample DNA into a qPCR reaction tube, perform a qPCR reaction, and interpret the gene mutation site based on the measured CT value to determine the type of ABCC11 gene mutation.

7. The method of claim 6, characterized in that, Step 2) involves mixing the positive and negative control samples with the reagents in the kit, placing them in a qPCR reaction tube, performing the qPCR reaction, and measuring the CT value.

8. The method as described in claim 6 or 7, characterized in that, If, in step 2), the CT value of the qPCR reaction using the c.538G primer-probe combination in the kit is ≤35, and the CT value of the qPCR reaction using the c.538A primer-probe combination in the kit is >35, then the genotype of the sample being tested is GG, and the result is positive.

9. The method as described in claim 6 or 7, characterized in that, If, in step 2), the CT value of the qPCR reaction using the c.538G primer-probe combination in the kit is ≤35, and the CT value of the qPCR reaction using the c.538A primer-probe combination in the kit is ≤35, then the genotype of the sample being tested is GA, and the result is positive.

10. The method as described in claim 6 or 7, characterized in that, If, in step 2), the CT value of the qPCR reaction using the c.538G primer-probe combination in the kit is >35, and the CT value of the qPCR reaction using the c.538A primer-probe combination in the kit is ≤35, then the genotype of the sample being tested is AA, and the result is negative.

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