Method for detecting mutation of long-fragment nucleic acid molecules and application of method

Abstract

The invention relates to the technical field of gene detection, and particularly discloses a method for detecting mutation of long-fragment nucleic acid molecules and application of the method. According to the method disclosed by the invention, the target gene is subjected to segmented amplification and then is subjected to nanopore sequencing, so that complete and more comprehensive gene data is obtained, and more comprehensive and more accurate diagnosis or analysis results can be provided.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, and in particular to a method for detecting long-fragment nucleic acid molecular mutations and its application. Background Technology

[0002] Cystic fibrosis (CF) is a common autosomal recessive lethal genetic disorder. Its pathogenesis stems from a biallelic variation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR The gene is located in the 7q31.2 chromosome region, is approximately 230 kb in length, and contains 27 exons. Most CF patients typically present with multi-organ disease. CF has a high incidence rate and often results in a high early mortality rate due to respiratory failure. To date, more than 2,000 gene variants that can lead to CF and cause abnormal function of the CFTR protein have been identified. Given the high prevalence of CF, accurate detection of CFTR gene variants is crucial for assessing reproductive risk, guiding patient treatment, and developing new drugs.

[0003] It is worth noting that data from the Cystic Fibrosis Foundation Patient Registry (CFFPR) in 2018 showed that approximately 3.1% of CF patients had fewer than two tests. CFTR Genetic variations indicate that current testing methods cannot detect all variations. Precise detection is needed. CFTR Genetic testing and typing are not only crucial for diagnostic accuracy but also directly impact clinical decision-making regarding CF treatment drugs. Furthermore, complete... CFTR Genetic testing data can also help establish genotype-phenotype associations and provide data support.

[0004] Existing detection technologies for CFTR each have their own characteristics: While dideoxy termination sequencing (Sanger sequencing) is the gold standard for detecting point mutations in exon regions, it has throughput limitations and is costly and time-consuming. Multiplex ligation-dependent probe amplification (MLPA) specializes in detecting long-fragment variants, but it determines variants based on relative fluorescence, making it susceptible to interference from experimental procedures and environmental factors, thus compromising accuracy. High-throughput sequencing (NGS) is suitable for detecting shorter fragments and has limitations in detecting large deletions and rearrangements.

[0005] Therefore, there is an urgent need for a method that can overcome the above problems and achieve more comprehensive results quickly, with high throughput and low cost. CFTR Methods for detecting genetic information. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a CFTR gene detection method based on nanopore sequencing and multi-fragment amplification, and its application. This invention is based on nanopore sequencing technology and detects CFTR genes through long-fragment multiplex amplification. CFTR This invention provides information on variations in all exons and most introns of a gene. It is characterized by speed, high throughput, and low cost, making it suitable for large-scale applications. Furthermore, the method in this invention can obtain more comprehensive gene variation information, including point mutations, insertions, deletions, and large-segment rearrangements, thereby clarifying the phase relationships between variant sites, increasing the understanding of disease phenotype-genotype correlations, and facilitating accurate disease diagnosis.

[0007] In a first aspect, the present invention provides a method for detecting mutations in long nucleic acid molecules, comprising the following steps: (1) Divide the long-fragment nucleic acid molecules into amplicon regions and construct primer sets based on the divided regions; (2) Multiplex PCR amplification of the test samples was performed using primer sets to obtain amplification products; (3) Construct nanopore sequencing libraries based on amplification products, obtain sequencing data, and analyze the sequencing data to obtain long-fragment nucleic acid molecular mutation results.

[0008] In some embodiments of the present invention, the length of the long fragment nucleic acid molecule is not less than 84 kb.

[0009] In some embodiments of the present invention, the length of the long fragment nucleic acid molecule is 84-120 kb.

[0010] In some embodiments of the present invention, the length of the long fragment nucleic acid molecule is 84-108 kb.

[0011] In some embodiments of the present invention, the number of amplicon regions divided is 10-15.

[0012] In some embodiments of the present invention, the number of amplicon regions divided is 10-13.

[0013] In some embodiments of the present invention, the number of amplicon regions is 12.

[0014] In some embodiments of the present invention, the length of the amplicon is 7-15 kb.

[0015] In some embodiments of the present invention, the length of the amplicon is 7-10 kb.

[0016] In this invention, the number of amplicon regions and the length of the amplicon can be adjusted based on the actual situation of the target nucleic acid molecule, including but not limited to the ranges defined above.

[0017] In some embodiments of the present invention, the mutation results of the long-fragment nucleic acid molecules include at least one of the following: mutation location, amino acid residue substitution or base polymorphism and mutation frequency.

[0018] In some embodiments of the present invention, the mutation results of the long-fragment nucleic acid molecules include: point mutations, insertions and deletions, and rearrangements of large fragments.

[0019] In some embodiments of the present invention, the method further includes detecting mutations in multiple long-fragment nucleic acid molecules in the test sample.

[0020] In some embodiments of the present invention, the method is capable of simultaneously detecting mutations in multiple target long-fragment nucleic acid molecules in a test sample.

[0021] In some embodiments of the present invention, when simultaneously detecting mutations in multiple target long-fragment nucleic acid molecules in a test sample, the method includes: (1) Divide the amplicon regions of multiple long-fragment nucleic acid molecules and construct their respective primer sets according to the divided regions; (2) Multiplex PCR amplification was performed on the test samples using their respective primer sets to obtain amplification products of nucleic acid molecules of different lengths; (3) Nanopore sequencing libraries were constructed based on the amplification products of different long-fragment nucleic acid molecules, sequencing data was obtained, and the mutation results of long-fragment nucleic acid molecules were obtained by analyzing the sequencing data.

[0022] In some embodiments of the present invention, different tag sequences are ligated onto the amplification products of different long-fragment nucleic acid molecules during the construction of nanopore sequencing libraries.

[0023] In some embodiments of the present invention, the tag sequence includes, but is not limited to, barcode sequences, unique molecular identifiers (UMIs), HIS tag sequences, flag tag sequences, HA tag sequences, and V5 tag sequences. In this invention, any tag sequence capable of distinguishing different samples during sequencing falls within the scope of protection of tag sequences in this invention.

[0024] In some embodiments of the present invention, the tag sequence can be introduced directly by ligation, added to PCR amplification primers and added directly by amplification, or added in other ways. The present invention does not limit the method of introducing the tag sequence.

[0025] In some embodiments of the present invention, the length of the long nucleic acid fragment is limited as defined above.

[0026] In some embodiments of the present invention, the number of amplicon regions divided is limited as defined above.

[0027] In some embodiments of the present invention, the nucleic acid molecule includes at least one of DNA or RNA.

[0028] In some embodiments of the present invention, the long-fragment nucleic acid molecule includes CFTR The full-length sequence or a fragment thereof.

[0029] In some embodiments of the present invention, the long-fragment nucleic acid molecule includes CFTR All exon sequences.

[0030] In some embodiments of the present invention, the primer set includes: Selected from one group of SEQ ID NO: 1-2, SEQ ID NO: 3-4, and SEQ ID NO: 5-6. Selected from one group of SEQ ID NO: 7-8, SEQ ID NO: 9-10, and SEQ ID NO: 11-12. Selected from one of SEQ ID NO: 13-14 and SEQ ID NO: 15-16. Selected from one of SEQ ID NO: 17-18, SEQ ID NO: 19-20, and SEQ ID NO: 21-22. Selected from one of SEQ ID NO: 23-24 and SEQ ID NO: 25-26 Selected from one group of SEQ ID NO: 27-28, SEQ ID NO: 29-30, and SEQ ID NO: 31-32. Selected from one group of SEQ ID NO: 33-34, SEQ ID NO: 35-36, and SEQ ID NO: 37-38. Selected from one group of SEQ ID NO: 39-40, SEQ ID NO: 41-42, and SEQ ID NO: 43-44. Selected from one of SEQ ID NO: 45-46 and SEQ ID NO: 47-48. Selected from one group of SEQ ID NO: 49-50 and SEQ ID NO: 51-52. Selected from one group of SEQ ID NO: 53-54, SEQ ID NO: 55-56, and SEQ ID NO: 57-58, and Selected from one of SEQ ID NO: 59-60, SEQ ID NO: 61-62, and SEQ ID NO: 63-64.

[0031] In some embodiments of the present invention, the primer set is selected from one of the following combinations: (1) SEQ ID NO: 1-2, 11-12, 13-14, 17-18, 25-26, 27-28, 33-34, 39-40, 45-46, 49-50, 55-56 and 59-60; (2) SEQ ID NO: 1-2, 11-12, 13-14, 17-18, 25-26, 27-28, 33-34, 39-40, 45-46, 49-50, 57-58 and 59-60; (3) SEQ ID NO: 5-6, 11-12, 13-14, 21-22, 25-26, 31-32, 33-34, 43-44, 45-46, 49-50, 57-58 and 63-64.

[0032] In some embodiments of the present invention, the primer set is: SEQ ID NO: 5-6, 11-14, 21-22, 25-26, 31-34, 43-46, 49-50, 57-58 and 63-64.

[0033] In some embodiments of the present invention, SEQ ID NO: 5-6 targeted amplification CFTR The first exon.

[0034] In some embodiments of the present invention, SEQ ID NO: 11-12 targeted amplification CFTR The second and third exons.

[0035] In some embodiments of the present invention, SEQ ID NO: 13-14 targeted amplification CFTR The fourth to seventh exons.

[0036] In some embodiments of the present invention, SEQ ID NO: 21-22 targeted amplification CFTR The eighth to tenth exons.

[0037] In some embodiments of the present invention, SEQ ID NO: 25-26 targeted amplification CFTR The eleventh exon.

[0038] In some embodiments of the present invention, SEQ ID NO: 31-32 targeted amplification CFTR The twelfth to fourteenth exons.

[0039] In some embodiments of the present invention, SEQ ID NO: 33-34 targeted amplification CFTR Exons 15 through 17.

[0040] In some embodiments of the present invention, SEQ ID NO: 43-44 targeted amplification CFTR Exons 18 through 21.

[0041] In some embodiments of the present invention, SEQ ID NO: 45-46 targeted amplification CFTR The 22nd exon.

[0042] In some embodiments of the present invention, SEQ ID NO: 49-50 targeted amplification CFTR The 23rd exon.

[0043] In some embodiments of the present invention, SEQ ID NO: 57-58 targeted amplification CFTR The 24th exon.

[0044] In some embodiments of the present invention, SEQ ID NO: 63-64 targeted amplification CFTR Exons 25 to 27.

[0045] In some embodiments of the present invention, the construction of sequencing libraries can be performed using conventional methods in the art or commercially available kits.

[0046] In some embodiments of the present invention, the construction of the sequencing library includes: end repair and sequencing adapter ligation.

[0047] In some embodiments of the present invention, the construction of the sequencing library further includes the ligation of tag sequences. This step is used to distinguish samples; when only one nucleic acid molecule to be tested is present, tag sequence ligation may not be performed.

[0048] In some embodiments of the present invention, the construction of the sequencing library further includes the purification of the product.

[0049] In some embodiments of the present invention, the purification of the product can be performed using conventional methods in the art or commercially available kits, including but not limited to purification using commercially available magnetic beads.

[0050] In some embodiments of the present invention, the method specifically includes: (1) To CFTR The gene was divided into amplicon regions, comprising 11-13 amplicon regions, each 7-10 kb in length. These 11-13 amplicon regions cover… CFTR Primers were constructed for all exons of the gene, based on the defined regions. (2) Use primer sets to perform multiplex PCR amplification of the nucleic acid to be tested in the test sample to obtain amplification products; (3) Connect nanopore sequencing adapters to the amplification products to obtain nanopore sequencing libraries; (4) Sequencing data was obtained by sequencing the nanopore sequencing library, and the sequencing data was analyzed to obtain... CFTR Gene mutation results.

[0051] In a second aspect, the present invention provides a long-fragment nucleic acid molecular mutation detection product, the long-fragment nucleic acid molecular mutation detection product comprising: a specific primer set and a sequencing library construction reagent.

[0052] In some embodiments of the present invention, the amplified fragment of the specific primer set contains a complete nucleic acid molecule to be tested after ligation.

[0053] In some embodiments of the present invention, the specific primer set contains 10-15 primer pairs per nucleic acid molecule to be tested.

[0054] In some embodiments of the present invention, the long nucleic acid fragment molecules are defined as described above.

[0055] In some embodiments of the present invention, the specific primer set is defined as described above.

[0056] In some embodiments of the present invention, the sequencing library construction reagent includes at least one of: end repair reagent or sequencing adapter reagent.

[0057] In some embodiments of the present invention, the sequencing library construction reagent further includes: a tag sequence reagent.

[0058] In some embodiments of the present invention, the tag sequence is limited as defined above.

[0059] In some embodiments of the present invention, the long-fragment nucleic acid molecule includes CFTR The full-length sequence or a fragment thereof.

[0060] A third aspect of the invention provides the use of the long-fragment nucleic acid molecular mutation detection product described above in the preparation of products for diagnosing cystic fibrosis (CF).

[0061] The beneficial effects of this invention are: This invention provides a method for detecting [the following] based on nanopore sequencing technology combined with long fragment amplification. CFTR A method for detecting variations in all exons and most introns of a gene. This method is simple to operate and facilitates its widespread application.

[0062] The detection method in this invention can obtain more comprehensive information on target gene variations, including point mutations, insertions, deletions, and large-fragment rearrangements. Furthermore, it can clarify the phase relationship between variation sites, increase the understanding of the correlation between disease phenotype and genotype, and facilitate the correct diagnosis of the disease, thereby providing more comprehensive and accurate CF detection results.

[0063] The detection method in this invention combines nanopore sequencing technology, which is fast, high-throughput, and low-cost, and can be used for large-scale detection needs. Attached Figure Description

[0064] Figure 1 For the method in the embodiments of the present invention CFTR A schematic diagram showing the primer amplification region division of the 27 exons of a gene.

[0065] Figure 2 The results show the amplification of 12 regions using different primers.

[0066] Figure 3 The results show the multiplex PCR amplification of five different primer combinations (primer mixtures 1-5).

[0067] Figure 4 The sequencing results of PCR amplification products from primer mixture 1 (a), primer mixture 2 (b), and primer mixture 4 (c) are shown.

[0068] Figure 5 The sequencing results of PCR amplification products from four different primer concentration systems are shown, where abcd correspond to systems 1-4, respectively.

[0069] Figure 6 The sequencing results of 5 known samples were obtained using the method described in this embodiment of the invention. Detailed Implementation

[0070] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0071] Example 1 This embodiment designed, screened, and obtained a set of methods for... CFTR The primer set for full-length gene amplification, and the specific experimental protocol are as follows: CFTR The gene contains 27 exons, which are divided into 12 regions. Two to three primer pairs are designed for each region, requiring amplicon length of 7-10 kb, and the resulting 12 amplicones to cover all exon regions. A schematic diagram of the 27 exon region division is shown below. Figure 1 As shown in the table below, the primers designed for each region are presented.

[0072] Table 1 CFTR Gene PCR amplification primer sequence information

[0073] Each primer pair was individually amplified by PCR, and the amplification system is shown in the table below.

[0074] Table 2 PCR amplification system (10 μL)

[0075] The amplification program was as follows: 94℃ for 1 min; 98℃ for 10 s, 60℃ for 15 s, 68℃ for 8 min, for 25 cycles; 68℃ for 2 min; and incubation at 4℃. After amplification, approximately 2 μL of the PCR product was subjected to 0.6% agarose gel electrophoresis, and the optimal primers were determined based on the electrophoresis results.

[0076] The results of amplification of 12 regions using different primers individually are as follows: Figure 2 As shown.

[0077] It can be observed that all primers can obtain the target product. Specifically, regions 1, 4, 6, and 12 each have 3 primer pairs with good specificity and almost no non-specific amplification. Regions 3, 7, 8, 10, and 11 each have 2 primer pairs with good specificity and almost no non-specific amplification. Region 2 has only 1 primer pair that can specifically amplify the product, while the other 2 primer pairs produce a large amount of non-specific amplification products. Primers in regions 5 and 9 almost all exhibit some degree of non-specific amplification, but CFTR-5_2 and CFTR-9_1 produce relatively fewer non-specific products compared to the others.

[0078] Example 2 A multiplex amplification system composed of primers with good specificity is more conducive to the detection of target sites compared with existing amplification methods. Therefore, in this embodiment, a multiplex amplification system was constructed using primer combinations that have been verified to have good individual amplification effects in Example 1, and primers suitable for multiplex amplification were screened.

[0079] The specific experimental method is as follows: (1) Construction of the multiplex amplification system and preliminary screening of multiplex amplification primer combinations Based on the results in Example 1, a total of 5 combinations of multiplex amplification primers (primer mixtures) were constructed, and the specific combinations are shown in the table below.

[0080] Table 3 Primer combinations for multiplex amplification (primer mixtures)

[0081] Based on the above five combinations of multiplex amplification primers, a multiplex PCR amplification system was constructed.

[0082] Table 4. Multiplex PCR amplification system (50 μL)

[0083] The amplification program was as follows: 94℃ for 1 min; 98℃ for 10 s, 60℃ for 15 s, 68℃ for 4 min, repeated 25 times; 68℃ for 2 min; and incubated at 4℃. The PCR amplification product was obtained. 40 μL of the PCR amplification product was purified using 0.4× magnetic beads.

[0084] Approximately 100 ng of PCR amplification products before and after purification with magnetic beads were subjected to 0.6% agarose gel electrophoresis. Based on the electrophoresis results, primer combinations with good multiplex amplification performance were initially selected.

[0085] The results are as follows Figure 3 As shown.

[0086] It was observed that the amplification products of all five primer mixtures contained non-specific amplification products <3 kb. However, after purification with magnetic beads, the non-specific amplification products of primer mixtures 1 and 2 were effectively removed, while the amplification product of primer mixture 4 still contained non-specific amplification products after purification. Furthermore, the amplification products of primer mixtures 3 and 5 showed obvious non-specific amplification bands, especially the ~3 kb non-specific products, which were difficult to remove by magnetic bead purification.

[0087] Therefore, primer mixtures 1, 2, and 4 will be used in subsequent experiments to further screen the primer mixtures.

[0088] (2) Construction of sequencing libraries and secondary screening of multiplex amplification primer combinations: The presence of a large number of nonspecific products in the multiplex PCR amplification system will reduce the amount of effective data. Although the above steps have performed a preliminary screening of the multiplex amplification primer combinations, the agarose gel electrophoresis results cannot directly reflect the actual detection of the 12 amplicons. Therefore, library construction and sequencing are still required to determine the actual detection of each amplicon and to perform a secondary screening of the multiplex amplification primer combinations.

[0089] In this embodiment, the construction of the sequencing library is achieved based on the CycloneSEQ 24 barcode library preparation kit (CycloneSEQ).

[0090] The specific steps for constructing a sequencing library are as follows: The PCR amplification products obtained from multiplex primer combinations were purified using 0.4× magnetic beads and then subjected to end repair. The end repair reaction solution (60 μL) consisted of: 1 μg of purified PCR amplification product, 12 μL of end repair buffer, and 3 μL of end repair enzyme, with nuclease-free water added to bring the total volume to 60 μL. The end repair reaction program was as follows: incubation at 20°C for 10 min, followed by incubation at 65°C for 10 min to obtain the end-repaired product. The end-repaired product was then purified using 1× magnetic beads.

[0091] The purified end-repair products were barcoded, and the barcode linking system is shown in the table below.

[0092] Table 5. Barcode Linking System (40 μL)

[0093] After reacting at 25℃ for 30 min, the product was purified using 0.4× magnetic beads to obtain the purified barcode ligation product.

[0094] The purified barcode ligation products of the three primer mixtures were mixed in equal masses and then ligated into nanopore sequencing adapters. The nanopore sequencing adapter ligation system was prepared according to the table below.

[0095] Table 6. Nanopore sequencing adapter ligation system (400 μL)

[0096] In this embodiment, the purified barcode ligation product was 31.7 μL.

[0097] After reacting at 25℃ for 30 min, the mixture was purified using 0.4× magnetic beads to obtain purified nanopore sequencing adapter ligation products, i.e., nanopore sequencing libraries.

[0098] The obtained nanopore sequencing libraries were sequenced using the CycloneSEQ WT sequencing kit (CycloneSEQ), the CycloneSEQ WT sequencing chip (CycloneSEQ), and its compatible nanopore gene sequencer CycloneSEQ-WT02 (CycloneSEQ). 900 ng of nanopore sequencing library was loaded into each sequencing chip, and sequencing was performed at 30°C.

[0099] The results are as follows Figure 4 As shown.

[0100] It was found that all three primer mixtures could effectively detect 12 amplicones. Among them, the sequencing depth of the 12 amplicones from primer mixtures 1 and 2 differed significantly, especially the dominant amplification in region 1. In contrast, the sequencing depth of the 12 amplicones from primer mixture 4 showed relatively smaller differences, which is more conducive to rapid adjustment of the system balance in the subsequent process.

[0101] Therefore, primer mixture 4 was used as the optimal primer mixture for optimizing the subsequent multiplex amplification system.

[0102] Example 3 In this embodiment, primer mixture 4 was used as the optimal primer mixture to investigate the effect of primer concentration on the final detection effect in the multiplex PCR amplification system.

[0103] The specific experimental method is as follows: Using the human genomic DNA standard NA12878 as a template, PCR amplification, library construction, and sequencing were performed according to the methods described in the above embodiments. Primer mixture 4 was used as a primer mixture for multiplex PCR amplification. The difference was that the concentration of primers in each region of primer mixture 4 was adjusted, resulting in four PCR multiplex amplification systems.

[0104] The concentration adjustments of primers in each region of the four PCR multiplex amplification systems are shown in the table below.

[0105] Table 7. System concentrations of primers for each region in four PCR multiplex amplification systems.

[0106] After sequencing was completed, the sequencing depth of each amplicon was analyzed.

[0107] The results are as follows Figure 5 As shown.

[0108] It can be observed that in PCR multiplex amplification systems 1 and 2, three regions showed low sequencing depths of the amplicon sequences. System 3 exhibited significant differences in sequencing depth among the amplicon sequences, which was detrimental to the final detection results. Although the sequencing depth in region 3 of system 4 was slightly higher, the overall sequencing depth of the amplicon sequences was relatively balanced. Therefore, considering the overall balance of the systems and the clear detection of loci, system 4 was selected for detection. CFTR The final multiplex amplification system of genes.

[0109] Example 4 In this embodiment, the system based on system 4 was verified. CFTR The effectiveness and reliability of full-length gene detection methods.

[0110] The specific verification method is as follows: Based on the results of Example 3, System 4 was used to test 5 known samples to verify the accuracy of the detection effect. The multiplex PCR amplification, library construction and sequencing methods were the same as those in the above examples.

[0111] The results are shown in Table 8 and Figure 6 As shown.

[0112] Table 8. Detection results of 5 known samples

[0113] The results above show that the method in this embodiment of the invention can accurately detect the status of each sample (including mutation status and mutation frequency) with an accuracy of 100%. Moreover, the sequencing balance among samples is good, and the data availability is good.

[0114] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting long-fragment nucleic acid molecular mutations, characterized in that, The method includes the following steps: (1) Divide the long-fragment nucleic acid molecules into amplicon regions and construct primer sets based on the divided regions; (2) Multiplex PCR amplification of the test samples was performed using primer sets to obtain amplification products; (3) Construct nanopore sequencing libraries based on amplification products, obtain sequencing data, and analyze the sequencing data to obtain long-fragment nucleic acid molecular mutation results; The length of the long-fragment nucleic acid molecule is not less than 84 kb; The number of amplicon regions is 10-15.

2. The method according to claim 1, characterized in that, The length of the amplicon is 7-15 kb.

3. The method according to claim 1, characterized in that, The results of mutations in long-fragment nucleic acid molecules include at least one of the following: mutation location, amino acid residue substitution or base polymorphism, and mutation frequency.

4. The method according to claim 1, characterized in that, The method also includes detecting mutations in multiple long-fragment nucleic acid molecules in the test sample.

5. The method according to claim 4, characterized in that, The method includes: (1) Divide the amplicon regions of multiple long-fragment nucleic acid molecules and construct their respective primer sets according to the divided regions; (2) Multiplex PCR amplification was performed on the test samples using their respective primer sets to obtain amplification products of nucleic acid molecules of different lengths; (3) Nanopore sequencing libraries were constructed based on the amplification products of different long-fragment nucleic acid molecules, sequencing data was obtained, and the mutation results of long-fragment nucleic acid molecules were obtained by analyzing the sequencing data; In the process of constructing nanopore sequencing libraries, different tag sequences are linked to the amplification products of different long-fragment nucleic acid molecules; The length of each long-fragment nucleic acid molecule is not less than 84 kb; The number of amplicon regions divided is 10-15.

6. The method according to any one of claims 1-5, characterized in that, The nucleic acid molecule includes at least one of DNA or RNA.

7. The method according to any one of claims 1-5, characterized in that, The long-fragment nucleic acid molecules include CFTR The full-length sequence or a fragment thereof; The primer set includes: Selected from one group of SEQ ID NO: 1-2, SEQ ID NO: 3-4, and SEQ ID NO: 5-6. Selected from one group of SEQ ID NO: 7-8, SEQ ID NO: 9-10, and SEQ ID NO: 11-12. Selected from one of SEQ ID NO: 13-14 and SEQ ID NO: 15-16. Selected from one of SEQ ID NO: 17-18, SEQ ID NO: 19-20, and SEQ ID NO: 21-22. Selected from one of SEQ ID NO: 23-24 and SEQ ID NO: 25-26 Selected from one group of SEQ ID NO: 27-28, SEQ ID NO: 29-30, and SEQ ID NO: 31-32. Selected from one group of SEQ ID NO: 33-34, SEQ ID NO: 35-36, and SEQ ID NO: 37-38. Selected from one group of SEQ ID NO: 39-40, SEQ ID NO: 41-42, and SEQ ID NO: 43-44. Selected from one of SEQ ID NO: 45-46 and SEQ ID NO: 47-48. Selected from one group of SEQ ID NO: 49-50 and SEQ ID NO: 51-52. Selected from one group of SEQ ID NO: 53-54, SEQ ID NO: 55-56, and SEQ ID NO: 57-58, and Selected from one of SEQ ID NO: 59-60, SEQ ID NO: 61-62, and SEQ ID NO: 63-64.

8. The method according to claim 7, characterized in that, The primer set is selected from one of the following combinations: (1) SEQ ID NO: 1-2, 11-12, 13-14, 17-18, 25-26, 27-28, 33-34, 39-40, 45-46, 49-50, 55-56 and 59-60; (2) SEQ ID NO: 1-2, 11-12, 13-14, 17-18, 25-26, 27-28, 33-34, 39-40, 45-46, 49-50, 57-58 and 59-60; (3) SEQ ID NO: 5-6, 11-12, 13-14, 21-22, 25-26, 31-32, 33-34, 43-44, 45-46, 49-50, 57-58 and 63-64.

9. A long-fragment nucleic acid molecular mutation detection product, characterized in that, The long-fragment nucleic acid molecular mutation detection products include: Specific primer set and sequencing library construction reagents; The amplified fragments from the specific primer set, after ligation, contain complete nucleic acid molecules to be tested. The specific primer set contains 10-15 primer pairs per nucleic acid molecule to be tested; The long-fragment nucleic acid molecules include CFTR The full-length sequence or a fragment thereof; The specific primer set includes: SEQ ID NO: 5-6, 11-14, 21-22, 25-26, 31-34, 43-46, 49-50, 57-58 and 63-64.

10. Use of the long-fragment nucleic acid molecular mutation detection product of claim 9 in the preparation of products for diagnosing cystic fibrosis (CF).

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