CFTR gene mutation combination, amplification reagent and application of product in preparation of CF risk assessment product

Abstract

The invention belongs to the technical field of gene detection, and particularly relates to application of a CFTR gene mutation combination, an amplification reagent and a product in preparation of a CF risk assessment product. The CFTR gene mutation combination consists of an intron region mutation combination and an exon region mutation combination, based on the CFTR gene mutation combination, the invention further develops an amplification reagent and a CFTR gene mutation detection product, the amplification reagent and the CFTR gene mutation detection product comprise a primer combination for multiplex PCR amplification of the CFTR gene mutation combination, and the primer combination has the characteristics of high accuracy, high specificity and high sensitivity, can accurately detect related gene mutation, and provides a reliable basis for risk assessment and diagnosis of CF. When the amplification reagent and the CFTR gene mutation detection product are combined with sweat chlorine detection for use, the diagnosis rate of CF can be remarkably improved, and the amplification reagent has important application value in clinical diagnosis of CF and is expected to provide more powerful support for early diagnosis and treatment of CF patients.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene detection, and particularly relates to application of a CFTR gene mutation combination, an amplification reagent and a product in preparation of a CF risk assessment product. BACKGROUND

[0002] Cystic fibrosis (CF) is an autosomal recessive genetic disease, and its pathogenesis is that a CFTR gene mutation on the long arm of chromosome 7 causes abnormal structure and function of CFTR protein, destroys the transport of chloride ions and water molecules in and out of the cell membrane, and makes the mucus secreted by the exocrine glands of the body thick, which blocks the lumen of organs such as the airway and the digestive tract and affects their function. In terms of clinical manifestations, patients often have chronic cough, sputum and thick sputum that is difficult to cough out, easy to cause repeated lung infection, and further cause bronchiectasis, atelectasis and even respiratory failure; the exocrine pancreatic insufficiency of the digestive system can cause fatty diarrhea, malnutrition, and may be accompanied by intestinal obstruction and rectal prolapse, and liver involvement can cause biliary cirrhosis; in addition, the concentration of chloride ions and sodium ions in the sweat of patients increases, and the patient is prone to electrolyte imbalance in high temperature, and male patients are often infertile due to vas deferens obstruction.

[0003] Studies have shown that the clinical characteristics and gene lineage of CF patients in different countries are different: the most common typical manifestation of Chinese CF patients is bronchiectasis, while the typical symptoms of exocrine pancreatic insufficiency in European and American CF patients are less common in Chinese patients. Therefore, many Chinese CF patients are initially misdiagnosed as non-cystic fibrosis bronchiectasis, chronic pneumonia or asthma and other diseases. At present, the domestic CF detection and diagnosis technology still has limitations. The existing CFTR mutation detection scheme based on the European and American population design is difficult to meet the specific mutation detection needs of the Chinese population because it only covers common mutation sites; and the fixed threshold standard of sweat chloride detection used in the diagnosis process cannot accurately adapt to the clinical characteristics of Chinese patients. The limitations of such detection methods and diagnostic standards result in a large number of missed diagnoses and misdiagnoses in the high-risk population of Chinese CF patients, especially in the bronchiectasis patient group.

[0004] Therefore, it is an urgent need to develop CF screening and diagnosis technology targeting the characteristics of the Chinese population, covering a wider range of gene mutation sites, combined with the application of standardized sweat chloride testing, to improve the accuracy of early diagnosis of CF in China. SUMMARY

[0005] In view of the above deficiencies, the application provides application of a CFTR gene mutation combination, an amplification reagent and a product in preparation of a CF risk assessment product.

[0006] The technical scheme of the application is as follows: In one aspect, the present application provides a CFTR gene mutation combination, which consists of an intron region mutation combination and an exon region mutation combination. The intron region mutation combination comprises: c.164+12T>C, c.869+3A>T, c.1210-11T>G, c.1766+5G>T, c.1767-16T>C, c.3717+45G>A, c.3874-4522A>G and c.3874-25C>A. The exon region mutation combination comprises: c.202A>G, c.263T>G, c.326A>G, c.437A>C, c.601G>A, c.650A>G, c.1069G>A, c.1251C>A, c.1265C>T, c.1666A>G, c.1950C>A, c.2083_2084insG, c.2812G>T, c.2909G>A, c.3289C>T, c.3068T>G, c.3746G>T, c.4056G>C, c.4297G>A and c.4357C>T.

[0007] In another aspect, the present application provides a primer combination for amplifying the CFTR gene mutation combination.

[0008] Specifically, the nucleotide sequence of the primer combination is shown in SEQ ID NO. 1-40.

[0009] Preferably, in the CFTR gene mutation combination: The nucleotide sequence of the forward primer of c.164+12T>C is shown in SEQ ID NO. 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 2; The nucleotide sequence of the forward primer of c.869+3A>T is shown in SEQ ID NO. 3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 4; The nucleotide sequence of the forward primer of c.1210-11T>G is shown in SEQ ID NO. 5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 6; The nucleotide sequence of the forward primer of c.1766+5G>T or c.1767-16T>C is shown in SEQ ID NO. 7, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 8; The nucleotide sequence of the forward primer of c.3717+45G>A is shown in SEQ ID NO. 9, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 10; The nucleotide sequence of the forward primer of c.3874-4522A>G or c.3874-25C>A is shown as SEQ ID NO. 11, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 12; The nucleotide sequence of the forward primer of c.202A>G or c.263T>G is shown as SEQ ID NO. 13, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 14; The nucleotide sequence of the forward primer of c.326A>G or c.437A>C is shown as SEQ ID NO. 15, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 16; The nucleotide sequence of the forward primer of c.601G>A or c.650A>G is shown as SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 18; The nucleotide sequence of the forward primer of c.1069G>A is shown as SEQ ID NO. 19, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 20; The nucleotide sequence of the forward primer of c.1251C>A or c.1265C>T is shown as SEQ ID NO. 21, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 22; The nucleotide sequence of the forward primer of c.1666A>G is shown as SEQ ID NO. 23, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 24; The nucleotide sequence of the forward primer of c.1950C>A or c.2083_2084insG is shown as SEQ ID NO. 25, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 26; The nucleotide sequence of the forward primer of c.2812G>T is shown as SEQ ID NO. 27, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 28; The nucleotide sequence of the forward primer of c.2909G>A is shown as SEQ ID NO. 29, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 30; The nucleotide sequence of the forward primer of c.3289C>T or c.3068T>G is shown as SEQ ID NO. 31, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 32; The nucleotide sequence of the forward primer of c.3746G>T is shown as SEQ ID NO. 33, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 34; The nucleotide sequence of the forward primer of c.4056G>C is shown as SEQ ID NO. 35, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 36; The nucleotide sequence of the forward primer of c.4297G>A is shown as SEQ ID NO. 37, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 38; The nucleotide sequence of the forward primer of c.4357C>T is shown as SEQ ID NO. 39, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 40.

[0010] In another aspect, the present application provides an amplification reagent, which comprises the primer combination of any one of the above.

[0011] Specifically, the final concentration of each primer in the amplification reagent is 50-200 nM.

[0012] Preferably, the final concentration of each primer in the amplification reagent is 100 nM.

[0013] In another aspect, the present application provides the use of the CFTR gene mutation combination, the primer combination or the amplification reagent of any one of the above in the preparation of a product.

[0014] Preferably, the product comprises: a CFTR gene mutation detection product and / or a CFTR gene mutation detection product.

[0015] Preferably, the type of the product comprises a chip and / or a kit.

[0016] In another aspect, the present application provides a CFTR gene mutation detection product, which comprises the CFTR gene mutation combination, the primer combination or the amplification reagent of any one of the above.

[0017] Preferably, the CFTR gene mutation detection product further comprises one or more of DNA polymerase, dNTPs, PCR reaction buffer, and negative quality control.

[0018] Specifically, the use method of the CFTR gene mutation detection product comprises the following steps: S1, extracting a DNA sample; S2, performing PCR amplification on the CFTR gene mutation combination by using the primer combination or the amplification reagent; S3, the amplified product is analyzed by hybridization or sequencing technology to determine whether a CFTR gene mutation exists.

[0019] Preferably, the DNA sample in step S1 is extracted from blood.

[0020] Preferably, the CFTR gene mutation in step S3 includes any one or more of c.164+12T>C, c.869+3A>T, c.1210-11T>G, c.1766+5G>T, c.1767-16T>C, c.3717+45G>A, c.3874-4522A>G and c.3874-25C>A, c.202A>G, c.263T>G, c.326A>G, c.437A>C, c.601G>A, c.650A>G, c.1069G>A, c.1251C>A, c.1265C>T, c.1666A>G, c.1950C>A, c.2083_2084insG, c.2812G>T, c.2909G>A, c.3289C>T, c.3068T>G, c.3746G>T, c.4056G>C, c.4297G>A, c.4357C>T.

[0021] In another aspect, the present application provides a product for CF risk assessment or diagnosis, which comprises the above-mentioned CFTR gene mutation combination, the above-mentioned primer combination, amplification reagent or CFTR gene mutation detection product.

[0022] Specifically, the product is used for CF risk assessment or diagnosis according to the CFTR gene mutation detection result.

[0023] Preferably, the standard for CF risk assessment or diagnosis is that a CFTR gene mutation is detected.

[0024] Further preferably, the CFTR gene mutation comprises any one or more of: c.164+12T>C, c.869+3A>T, c.1210-11T>G, c.1766+5G>T, c.1767-16T>C, c.3717+45G>A, c.3874-4522A>G and c.3874-25C>A, c.202A>G, c.263T>G, c.326A>G, c.437A>C, c.601G>A, c.650A>G, c.1069G>A, c.1251C>A, c.1265C>T, c.1666A>G, c.1950C>A, c.2083_2084insG, c.2812G>T, c.2909G>A, c.3289C>T, c.3068T>G, c.3746G>T, c.4056G>C, c.4297G>A, c.4357C>T.

[0025] In particular, the product can be used in combination with a sweat chloride concentration detection method for CF risk assessment or diagnosis.

[0026] Preferably, the sweat chloride concentration detection method is used for CF risk assessment according to the sweat chloride concentration.

[0027] Further preferably, the CF risk assessment criteria are: sweat chloride concentration ≥ 60 mmol / L, assessed as high risk of CF; sweat chloride concentration of 30-59 mmol / L, assessed as medium risk of CF; and sweat chloride concentration < 30, assessed as low risk of CF.

[0028] Further preferably, the sweat chloride concentration detection method comprises any one or more of: coulometric analysis, colorimetry, ion selective electrode method, ion chromatography, enzyme method, flame photometry, potentiometric titration method, microfluidic chip method, and mass spectrometry.

[0029] In another aspect, the application provides a method for CF risk assessment or diagnosis, which comprises using the CFTR gene mutation combination, the primer combination, the amplification reagent, the CFTR gene mutation detection product, or the product for CF risk assessment or diagnosis described above.

[0030] In particular, the method can be used in combination with other CF risk assessment methods for CF risk assessment or diagnosis.

[0031] Preferably, the other CF risk assessment method is a sweat chloride concentration detection method.

[0032] In particular, the method is suitable for Chinese patients with bronchiectasis.

[0033] Preferably, the population of the method includes any one or more of the following: (1) a teenager with onset or a child with repeated lower respiratory tract infections; (2) a patient with bronchiectasis accompanied by typical CF manifestations, such as repeated sinusitis, gastrointestinal symptoms, azoospermia, or clubbing; (3) severe bronchiectasis or chronic coughing and wheezing of unknown origin. The application of the method to such patients can perform early screening and diagnosis, so as to timely develop interventions such as nutritional support, sputum discharge rehabilitation, and CFTR modulators, which is conducive to improving the prognosis and quality of life of patients.

[0034] The present application has the following advantages: (1) The present application provides a combination of CFTR gene mutations, and develops an amplification reagent and a CFTR gene mutation detection product based on the combination of CFTR gene mutations, which contains a primer combination for multiplex PCR amplification of the combination of CFTR gene mutations, has the characteristics of high accuracy, high specificity and high sensitivity, and can accurately detect the relevant gene mutations, providing a reliable basis for risk assessment and diagnosis of CF, and filling the gap of the existing European and American detection panels.

[0035] (2) The amplification reagent and the CFTR gene mutation detection product of the present application are used in combination with sweat chloride detection to construct a multi-factor complementary diagnosis path, so that the three types of patients with high / medium / low sweat chloride can be disposed accordingly, and the diagnosis rate of CF can be significantly improved, which has important application value in the clinical diagnosis of CF, and is expected to provide stronger support for the early diagnosis and treatment of CF patients.

[0036] (3) The present application standardizes the screening process from patient selection, detection execution to result analysis. It provides clear technical implementation steps, standardizes and operationalizes the screening work, and is convenient for popularization in clinical and research. The present application realizes systematic process, and realizes the unity of innovation and effectiveness. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The determination results of dataset 1.

[0038] Figure 2 The determination results of dataset 2.

[0039] Figure 3 The combined results analysis of dataset 1 and dataset 2. DETAILED DESCRIPTION

[0040] The application will be further clarified by the following examples. The examples are only a part of the application and are not used to limit the application. The experimental methods used in the following examples are conventional experiments. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0041] Example 1 CFTR gene mutation combination The CFTR gene mutation combination of the application is composed of an intron region mutation combination and an exon region mutation combination. The intron region mutation combination is: c.164+12T>C, c.869+3A>T, c.1210-11T>G, c.1766+5G>T, c.1767-16T>C, c.3717+45G>A, c.3874-4522A>G and c.3874-25C>A. The exon region mutation combination is: c.202A>G, c.263T>G, c.326A>G, c.437A>C, c.601G>A, c.650A>G, c.1069G>A, c.1251C>A, c.1265C>T, c.1666A>G, c.1950C>A, c.2083_2084insG, c.2812G>T, c.2909G>A, c.3289C>T, c.3068T>G, c.3746G>T, c.4056G>C, c.4297G>A and c.4357C>T. As shown in Table 1: Table 1

[0042] Example 2 Primer of CFTR gene mutation combination The primer information for amplifying the CFTR gene mutation combination of the application is shown in Table 2: Table 2

[0043] Example 3 CFTR gene mutation detection kit and its use method 1. Preparation of amplification reagent of CFTR gene mutation combination In the amplification reagent of the application, the final concentration of each primer is shown in Table 3: Table 3

[0044] 2. Extraction of sample DNA Extract the DNA of the sample to be tested.

[0045] 3. PCR amplification PCR amplification system is shown in Table 4, and PCR amplification procedure is shown in Table 5.

[0046] Table 4

[0047] Table 5

[0048] 4. Analysis of PCR amplification results The amplified products are analyzed by hybridization technique or sequencing technique to determine whether CFTR gene mutation exists.

[0049] Comparative Example 1 CFTR gene mutation detection kit The CFTR gene mutation detection kit of Comparative Example 1 is different from the CFTR gene mutation detection kit of Example 3 only in that the preparation of the amplification reagent of the CFTR gene mutation combination is different.

[0050] In the amplification reagent of Comparative Example 1, the final concentration of each primer is shown in Table 6: Table 6

[0051] Comparative Example 2 CFTR gene mutation detection kit The CFTR gene mutation detection kit of Comparative Example 2 is different from the CFTR gene mutation detection kit of Example 3 only in that the preparation of the amplification reagent of the CFTR gene mutation combination is different.

[0052] In the amplification reagent of Comparative Example 2, the final concentration of each primer is shown in Table 5: Table 5

[0053] Comparative Example 3 CFTR gene mutation detection kit The CFTR gene mutation detection kit of Comparative Example 3 is different from the CFTR gene mutation detection kit of Example 3 only in that the preparation of the amplification reagent of the CFTR gene mutation combination is different.

[0054] In the amplification reagent of Comparative Example 3, the final concentration of each primer is shown in Table 6: Table 6

[0055] Experimental Example 1 5 CFTR gene mutation detection kit accuracy, specificity and sensitivity verification 1. Accuracy and specificity verification of CFTR gene mutation detection kit Chinese patients who meet the imaging diagnostic criteria of bronchiectasis are selected, with special attention to those with CF suggestive clinical phenotypes. According to the Chinese CF diagnosis and treatment consensus, when chest CT shows that bronchiectasis mainly involves the upper lobe, or young patients with bronchiectasis, CF should be highly suspected, and patients who meet the conditions are included in the subsequent screening process. DNA is extracted from the peripheral blood samples of patients as CF positive samples. Healthy Chinese patients are selected, and DNA is extracted from the peripheral blood samples of patients as CF negative samples.

[0056] The CFTR gene mutation detection kit prepared by Example 3, Comparative Example 1-Comparative Example 3, respectively, and the method described in Example 3, the above CF positive samples and CF negative samples are detected. Verify the accuracy of the CFTR gene mutation detection kit.

[0057] The test results show that the CFTR gene mutation detection kit provided by Example 3 of the present application can accurately diagnose CF positive samples, with the highest accuracy. The CFTR gene mutation detection kit provided by Example 3 of the present application can accurately distinguish CF positive samples and CF negative samples, with high specificity.

[0058] Compared with the CFTR gene mutation detection kits of Comparative Examples 1-3, the CFTR gene mutation detection kit of Example 3 of the present application shows the best accuracy and specificity, and the above results confirm that the CFTR gene mutation combination of the present application has a synergistic effect.

[0059] 2. Sensitivity verification of CFTR gene mutation detection kit Samples containing all CFTR gene mutations are selected for mixing, and their genomic DNA is extracted as positive DNA, which is diluted to 1:50, 1:500, and 1:5000 samples.

[0060] The CFTR gene mutation detection kits prepared by Example 3, Comparative Example 1-Comparative Example 3 of the present application are used, and the method described in Example 3 is used to detect each concentration sample.

[0061] The test results show that the CFTR gene mutation detection kit provided by Example 3 of the present application has the highest sensitivity. Compared with Comparative Examples 1-3, the sensitivity is significantly improved.

[0062] Experimental Example 2 5 Further verification of accuracy and specificity of CFTR gene mutation detection kit In this example, the CFTR gene mutation detection kit prepared by Example 3 is further verified by the CFTR mutation data set.

[0063] Data set 1: A systematic review of the clinical and genetic characteristics of Chinese patients with cystic fibrosis, published in Pediatric pulmonology in 2020, searched for studies involving Chinese CF patients from January 1975 to August 2019. 88 CF patients with a clear diagnosis of CFTR mutation were identified, and 164 alleles were detected. The specific information of Dataset 1 is shown in Table 7: Table 7

[0064] Dataset 2: Genetic spectrum of Chinese children with cystic fibrosis: comprehensive data analysis from the main referral centre in China, published in Journal of medical genetics in 2022, characterized the genotypic features of Chinese children with CF, and summarized the detected CFTR spectrum of variations in Chinese CF patients (194 families, 388 alleles). The specific information of Dataset 2 is shown in Table 8: Table 8

[0065] The above datasets were verified using the CFTR gene mutation detection kit prepared in Example 3 according to the method described in Example 3.

[0066] The determination results of Dataset 1 are shown in Figure 1 The brown block in the figure represents the percentage of CFTR mutations that are not detected by the CFTR gene mutation detection kit of Example 3 of the present application, which is 63.41%. The sum of the remaining blocks is the detection rate of the CFTR gene mutation detection reagent of the present application, which is 36.59%.

[0067] The determination results of Dataset 2 are shown in Figure 2 The green block in the figure represents the percentage of CFTR mutations that are not detected by the CFTR gene mutation detection kit of Example 3 of the present application, which is 71.13%. The sum of the remaining blocks is the detection rate of the CFTR gene mutation detection reagent of the present application, which is 28.87%.

[0068] The sensitivity and specificity of the CFTR gene mutation detection kit of Example 3 of the present application were calculated in combination with Dataset 1 and Dataset 2. Sensitivity (%) = True positive / (True positive + False negative) x 100%; Specificity (%) = True negative / (True negative + False positive).

[0069] The true positive refers to the number of samples with CFTR mutations correctly detected by the kit; The false negative refers to the number of samples with CFTR mutations that the kit failed to detect; The true negative refers to the number of samples without CFTR mutations correctly determined by the kit; The false positive refers to the number of samples with CFTR mutations incorrectly determined by the kit.

[0070] The results show that the sensitivity of the CFTR gene mutation detection kit of Example 3 of the present application is 45.96%, and the specificity is 97.39%, as shown in Table 2. Figure 3 .

[0071] Experimental Example 3 CFTR gene mutation detection kit combined with sweat chloride detection 1. Patient inclusion criteria Chinese patients who meet the imaging diagnostic criteria for bronchiectasis were selected, with special attention to those with CF suggestive clinical phenotypes. According to the Chinese CF diagnosis and treatment consensus, patients with bronchiectasis who have cystic bronchiectasis as the main manifestation, involve multiple bronchiectasis, and have an early age of onset should be highly suspected of CF. Patients who meet the conditions are included in the subsequent screening process.

[0072] 2. Sweat chloride screening 2.1 Sweat collection Check if all the parts of the collection system are complete, connect the electrodes to the battery, the red one is the positive electrode and the black one is the negative electrode. Turn on the power and enter the information as prompted.

[0073] (1) Place the pilocarpine colloid on the electrode, use two pieces at a time, one for each positive and negative electrode. Clean the patient's test site and keep it dry, and use the electrode strap to bind it to the patient's arm. Generally, the red electrode (positive electrode) is bound to the front arm where there is more flesh, and the black electrode (negative electrode) should not be too far apart. (Note: The red electrode should be selected in a place with more flesh, which is rich in sweat glands, which is beneficial for sweat collection) (2) Connect the power supply and press the ">" button until the indicator light turns on, then start the pilocarpine import. After the import is complete, the circuit will automatically disconnect, remove the electrodes, and dispose of the two used colloid pieces, which cannot be reused. (Note: If the colloid cracks or shrinks significantly, it should not be used to avoid adverse consequences) (3) Simply wipe the red electrode introduction, using a disposable sweat collector to collect sweat, the collection time is generally in 20-30 minutes or so. (Note: collector must be firmly bound by the bandage in the patient's arm, except for special patients, the main reason that the sweat collection is not tight. If the collected sweat is not enough, repeat steps 3 and 4, but not recommended in the same part of the introduction of pilocarpine. (4) The collected sweat is transferred to the PCR tube, and the plastic film is sealed to ensure that the liquid is not volatile, ready for use. It can also be removed first, and then transferred to the PCR tube.

[0074] 2.2 Sweat collection considerations (1) The best forearm, upper arm or thigh can also be measured, if edema, the test cannot be performed.

[0075] (2) Do not use any chlorine-containing solution to clean the skin, do not use local anesthesia gel.

[0076] (3) Bilateral stimulation of sweat secretion can reduce the failure rate, while not increasing the collection time and causing discomfort to the subject.

[0077] (4) Unless the sweat collection or sweat test results are abnormal, a sweat collection can be performed.

[0078] (5) The average rate of sweat collection should not be less than 1 g / m2 / min, and samples with a rate lower than this cannot be analyzed. Insufficient sweat collection (<15 μL) cannot be analyzed, and the chloride content in samples with insufficient sweat collection is usually high.

[0079] (6) Liquid sweat collection specimens can be stored at 4°C in PCR tubes.

[0080] 2.3 Sweat chloride concentration determination (1) Install the three electrodes, install them in order on the instrument, red, black, three-hole electrode (2) Configure the working solution, add 20 drops of gelatin solution (SS-248GS) to a buffer (SS-248ABS), shake well, and introduce into a beaker for standby.

[0081] (3) Instrument calibration Place the beaker containing the working solution on the instrument, pull down the electrode, contact the working solution, and calibrate according to the instrument prompt.

[0082] (4) The sample size is 10 microliters, the instrument automatically tests, and the value is recorded.

[0083] (5) After the measurement is completed, the instrument must be cleaned immediately, especially the electrode, to prevent silver oxidation and affect subsequent use 2.4 Assessment of CF risk level in patients according to sweat chloride concentration The sweat chloride test is performed on the patients. The subjects are divided into three categories according to the sweat chloride concentration: ① High risk group: sweat chloride ≥ 60 mmol / L. This level directly suggests the possibility of CF, and the program automatically enters the genetic detection process; ② Medium risk group: sweat chloride between 30-59 mmol / L, which is a suspicious range. Genetic testing is required to determine the diagnosis; ③ Low risk group: sweat chloride < 30 mmol / L. Normal sweat chloride usually excludes CF. The multi-threshold screening system (≥ 60, 30-59, < 30) combined with clinical phenotype avoids missed diagnosis caused by a single sweat chloride threshold.

[0084] 3. Genetic testing The CFTR gene mutation detection kit and method of embodiment 3 of the present application are used for genetic testing of the high risk group and the medium risk group.

[0085] The test results show that the combination of sweat chloride detection and the CFTR gene mutation detection kit has good accuracy and specificity. It can effectively reduce missed diagnosis and misdiagnosis.

[0086] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not intended to limit the patent scope of the present application. It should be noted that any equivalent implementation or change that does not deviate from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the attached claims.

Claims

1. A combination of CFTR gene mutations, characterized in that, The CFTR gene mutation combination consists of an intron region mutation combination and an exon region mutation combination; The intron region mutation combination comprises: c.164+12T>C, c.869+3A>T, c.1210-11T>G, c.1766+5G>T, c.1767-16T>C, c.3717+45G>A, c.3874-4522A>G and c.3874-25C>A; The exon region mutation combination comprises: c.202A>G, c.263T>G, c.326A>G, c.437A>C, c.601G>A, c.650A>G, c.1069G>A, c.1251C>A, c.1265C>T, c.1666A>G, c.1950C>A, c.2083_2084insG, c.2812G>T, c.2909G>A, c.3289C>T, c.3068T>G, c.3746G>T, c.4056G>C, c.4297G>A and c.4357C>T.

2. A primer combination, characterized by The primer combination is used for amplifying the CFTR gene mutation combination in claim 1, and the nucleotide sequence of the primer combination is shown in SEQ ID NO. 1-40.

3. An amplification reagent, characterized by comprising The amplification reagent comprises the primer combination in claim 2, and the final concentration of each primer in the amplification reagent is 50-200 nM.

4. Use of the combination of mutations of the CFTR gene according to claim 1, of the combination of primers according to claim 2 or of the amplification reagents according to claim 3 for the preparation of a product, characterized in that, The product comprises: a CFTR gene mutation detection product and / or a CFTR gene mutation detection product; and the product type comprises: a chip and / or a kit.

5. A CFTR gene mutation detection product, characterized by, The CFTR gene mutation detection product comprises the CFTR gene mutation combination in claim 1, the primer combination in any one of claims 2 or the amplification reagent in claim 3.

6. The CFTR gene mutation detection product according to claim 5, characterized by, The use method of the CFTR gene mutation detection product comprises the following steps: S1, extracting a DNA sample; S2, performing PCR amplification on the CFTR gene mutation combination by using the primer combination or the amplification reagent; S3, performing analysis on the amplification product by using a hybridization technique or a sequencing technique to determine whether the CFTR gene mutation exists.

7. A product for CF risk assessment or diagnosis, characterized in that, The product comprises the CFTR gene mutation combination in claim 1, the primer combination in claim 2, the amplification reagent in claim 3 or the CFTR gene mutation detection product in any one of claims 5-6.

8. The product of claim 7, wherein, The product is used for CF risk assessment or diagnosis according to the CFTR gene mutation detection result; and the standard of the CF risk assessment or diagnosis is that the CFTR gene mutation is detected. The CFTR gene mutations include any one or more of c.164+12T>C, c.869+3A>T, c.1210-11T>G, c.1766+5G>T, c.1767-16T>C, c.3717+45G>A, c.3874-4522A>G and c.3874-25C>A, c.202A>G, c.263T>G, c.326A>G, c.437A>C, c.601G>A, c.650A>G, c.1069G>A, c.1251C>A, c.1265C>T, c.1666A>G, c.1950C>A, c.2083_2084insG, c.2812G>T, c.2909G>A, c.3289C>T, c.3068T>G, c.3746G>T, c.4056G>C, c.4297G>A, c.4357C>T.

9. The product of claim 7, wherein, The product can be used in combination with a sweat chloride concentration detection method for CF risk assessment or diagnosis; the sweat chloride concentration detection method is used for CF risk assessment according to the sweat chloride concentration.

10. The product of claim 9, wherein, The standard of the CF risk assessment is that the sweat chloride concentration is greater than or equal to 60 mmol / L, which is evaluated as CF high risk; the sweat chloride concentration is 30-59 mmol / L, which is evaluated as CF medium risk; and the sweat chloride concentration is less than 30, which is evaluated as CF low risk.

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