Detection method and application of XPV / POLH single base mutation

Abstract

The invention provides a detection method of XPV / POLH single base mutation and application of the detection method. The detection method comprises the following steps: by taking nucleic acid of a sample to be detected as template DNA, carrying out real-time fluorescence collection through PCR reaction, drawing an amplification curve and a melting curve, and carrying out result interpretation; the interpretation of the result comprises interpretation of mutation sites of four missense mutations and three nonsense mutations of the POLH gene. On the basis of a nucleic acid extraction method, detection of missense mutation and nonsense mutation of the POLH gene by using a melting curve is established, and the method has the advantages that operation is simple, operation can be carried out on the basis of PCR, consumed time is short, the whole process only needs about 2-3 hours, special equipment is not needed, and the method is suitable for large-scale popularization and application. A conventional fluorescent quantitative PCR instrument can realize interpretation of a detection result, the detection method is simple, the cost is low, the real-time fluorescent quantitative PCR method is equivalent, and a reliable and accurate basis can be quickly provided for auxiliary diagnosis of XP diseases.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a method and application for detecting XPV / POLH single-base mutations, and more particularly to a method for rapid extraction of peripheral blood genome and detection of XPV / POLH single-base mutations. Background Technology

[0002] Xeroderma pigmentosum (XP) is a rare skin disease, an autosomal recessive genetic disorder. Patients are sensitive to sunlight, and sun exposure may cause eye symptoms such as tearing and photophobia. Their skin is dry and rough, and sun-exposed areas are prone to freckle-like pigmentation or an increase in the number of freckle-like nevi, and may even experience skin atrophy. Some patients may also develop neurological disorders such as dementia or intellectual disability.

[0003] Studies have shown that some malignant tumors, such as melanoma and non-melanoma skin cancers, are associated with xeroderma pigmentosum syndrome. The pathogenesis of xeroderma pigmentosum involves changes in the body's structure and function caused by ultraviolet (UV) radiation. The normal human body counteracts UV damage by reducing these cumulative changes through nucleotide excision repair (NER), thereby reducing damage to the skin or nervous system and the risk of skin cancer. The XP gene plays a crucial role in NER. The increased sensitivity of xeroderma pigmentosum to UV radiation is closely related to its inability to counteract the formation of photoproducts in DNA through NER. The XP gene can be further subdivided into seven different subgroups called complementogenomes, including XP-A / XP-B / XP-C / XP-D / XP-E / XP-F / XP-G and one XP variant (XP-V). Different XP subtypes have different DNA excision repair deficiencies. When XPA-XPG and XPV are combined, they can work together to overcome UV-induced damage. Ultraviolet (UV) radiation induces the production of cyclobutanepyrimidine dimers (CPDs) / photoproducts and other chemically synthesized compounds, leading to DNA damage. XPCs and XPEs recognize photoproducts in DNA, XPBs and XPDs (which are part of the THIIH protein complex) open DNA structures at the photoproduct sites, XPA verifies the correct location of the THIIH protein, and XPGs and XPFs nucleases cleave damaged DNA structures, removing bad DNA fragments and replacing them with intact DNA. The XPV gene does not affect the NER pathway but participates in translesion synthesis (TLS). An XPV-encoded polymerase plays a crucial role in TLS; its absence increases the likelihood of mutations and cancer.

[0004] Clearly, defects in different XP genes lead to different consequences, and the clinical manifestations of XP patients vary depending on their genotype. All XP patients develop numerous freckle-like pigmented rashes on their skin when exposed to sunlight. Nearly 60% of patients are highly sensitive to sunlight; even short periods of strong sunlight or prolonged periods of weak sunlight can cause severe sunburn, blisters, and persistent erythema. For example, the XPC and XPE genotypes, and XPC and XPV patients, are prone to melanoma. Genotypes such as XPA, XPB, and XPD often lead to neurodegenerative diseases.

[0005] Furthermore, the skin symptoms of xeroderma pigmentosum are difficult to distinguish from other pigmentary disorders. Diagnosis based solely on phenotype leads to extremely high rates of missed and misdiagnosed cases. Even when a definitive diagnosis is made, the disease often progresses to the tumor stage. Clinical data indicates that XPV accounts for approximately 20% to 25% of all xeroderma pigmentosum patients. Moreover, because most XPV patients are not sensitive to sun exposure, they are often diagnosed only at the stage of skin tumors. Therefore, early diagnosis is of great significance in improving the prognosis of XPV patients.

[0006] Currently, genetic testing is widely used in the detection of various diseases, providing important evidence in areas such as disease diagnosis assistance, medication, and postoperative recovery monitoring. The diagnosis of xeroderma pigmentosum is based on clinical manifestations, family history, and / or identification of pathogenic variants in the XPA, XPB (ERCC3), XPC, XPD (ERCC2), XPE (DDB2), XPF (ERCC4), XPG (ERCC5), and XPV (POLH) genes. Genetic testing can assist in genetic counseling and prenatal diagnosis, early patient education, and appropriate protection, minimizing the harm of XP and improving prognosis.

[0007] In existing technologies, most XP gene detection is based on first- or second-generation sequencing methods. These sequencing methods have drawbacks: high requirements for clinical sample quality; time-consuming traditional nucleic acid extraction methods; the need for specialized equipment for automated nucleic acid extraction; long sequencing times; and the requirement for specialized personnel for data analysis. These characteristics make them inconvenient for clinicians and expensive, placing a heavy burden on patients. Chinese invention patent CN103540658A discloses a method, primers, and kit for detecting hotspot mutation sites in the human XPD gene. It targets the Lys751Gln mutation site in the xeroderma pigmentosum group D (XPD) gene, also known as the excision repair cross-complement gene, to improve the mutation detection rate. The combination of PCR and pyrosequencing technology can reduce false positive results due to the high sensitivity of PCR through direct confirmation of the gene sequence, thus improving the accuracy and specificity of the detection. However, this patented method can only detect the Lys751Gln mutation site in the ERCC2 gene and cannot identify all mutation sites.

[0008] Therefore, developing a simple, rapid, and accurate method for detecting single-base mutations in the XPV / POLH gene is of great clinical significance for the treatment of xeroderma pigmentosum. Summary of the Invention

[0009] To address the technical problems existing in the prior art, this invention provides a method and application for detecting single-base mutations in the XPV / POLH gene. The method is simple and rapid, and can detect single-base mutations in the XP gene, providing a more reliable experimental means for the clinical treatment of xeroderma pigmentosum.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting XPV / POLH single-base mutations, using the nucleic acid of the sample to be tested as template DNA, performing real-time fluorescence acquisition through PCR reaction, plotting amplification curves and melting curves, and interpreting the results; the interpretation of the results includes interpreting the mutation sites of 4 missense mutations and 3 nonsense mutations in the POLH gene.

[0011] In some embodiments, the PCR amplification reaction system includes a primer and probe composition, a PCR reaction solution, an enzyme solution, a positive control, and a negative control.

[0012] In some embodiments, the primer-probe composition includes a primer-probe combination for detecting non-mutated primers and a primer-probe combination for detecting mutations.

[0013] In some embodiments, the detection primer-probe combination introduces at least one artificial mutation site on the adjacent base at the 5' end of the mutation site to be detected and analyzed by the probe, and the base of the artificial mutation site is modified with locked nucleic acid.

[0014] In some embodiments, the primer-probe compositions are purified by HPLC.

[0015] In some embodiments, the primer-probe combination for detecting unmutated POLH genes includes primers and probes for detecting unmutated POLH genes.

[0016] In some embodiments, the mutation detection primer-probe combination includes primers and probes containing primers and probes for detecting mutations in the POLH gene.

[0017] In some embodiments, the sequences of the primers and probes in the unmutated primer-probe combination are shown in SEQ ID NO.:1, SEQ ID NO.:2, SEQ ID NO.:3, and SEQ ID NO.:4.

[0018] In some embodiments, the sequences of the primers and probes in the detection mutation primer-probe combination are as shown in SEQ ID NO.:5, SEQ ID NO.:6, SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:9, SEQ ID NO.:10, and SEQ ID NO.:11.

[0019] In some embodiments, the method for detecting XPV / POLH single-base mutations includes the following steps:

[0020] S1. Nucleic acid extraction: After centrifugation of peripheral blood samples, the leukocyte fluid is collected, added to leukocyte lysis buffer for lysis, and the supernatant is removed to obtain nucleic acid samples for later use;

[0021] S2. Reagent preparation: Take the probe primer mixture, PCR reaction solution, and enzyme solution, mix them evenly, and obtain the XPV melting curve mutation point detection solution;

[0022] S3. Amplification and detection: Take nucleic acid sample, XPV melting curve mutation point detection solution, negative control, wild-type positive control and mutant positive control, mix them to obtain PCR amplification reaction system, perform melting curve PCR amplification reaction, and perform real-time fluorescence quantitative detection on a fluorescence quantitative PCR instrument;

[0023] S4 plots PCR curves and melting curves, and interprets and analyzes the test results.

[0024] In some embodiments, in S1, the leukocyte lysis buffer is: 50 mM Tris-HCl pH 7.5, 150 mM KOH, 1 mM EDTA, 1.5% Tritonx-100, 1% Sodium deoxycholate, 0.2% SDS, 80 μg / ml proteinase K, and the volume is adjusted to 100 μL with physiological saline.

[0025] In some embodiments, the cell lysis buffer and leukocyte fluid are mixed at a volume ratio of 100:1, and then vortexed for 1 min to mix.

[0026] In some embodiments, in S3, the wild-type positive control is a TE buffer solution of a wild-type plasmid containing the POLH gene; the mutant positive control is a TE buffer solution of a mutant plasmid containing the POLH gene, wherein the mutation refers to a missense mutation in the POLH gene including mutation sites c.25G>T, c.126G>C, c.332G>A, c.364A>C, or a nonsense mutation including mutation sites c.490G>T, c.1066C>T, c.437dup.

[0027] In some embodiments, the amplified sequence of the wild-type plasmid is shown in SEQ ID NO.12.

[0028] In some embodiments, the amplified sequence of the mutant plasmid is shown in SEQ ID NO.13.

[0029] In some embodiments, the negative control is sterile water for injection.

[0030] In some embodiments, the concentration of wild-type plasmid in the wild-type positive control and the concentration of mutant plasmid in the mutant positive control are both 10. 6 ±5000 copies / mL.

[0031] In some embodiments, the amplification program of the PCR reaction is as follows: 95℃×2min; 95℃×10min, 60℃×1min (40 cycles); 95℃×10s, 55-85℃ (0.03℃ / s); with FAM and VIC fluorescence channels set at 60℃×1min and 60~90℃ for illumination.

[0032] In some embodiments, the method for interpreting the detection results includes:

[0033] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 68.9±0.5℃, it indicates that no mutation has occurred at the c.25G>T mutation site of the POLH gene.

[0034] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 66.6±0.5℃, it indicates that a mutation has occurred at the c.25G>T mutation site of the POLH gene.

[0035] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 75.1±0.5℃, it indicates that no mutation has occurred at the c.126G>C mutation site of the POLH gene.

[0036] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 72.8±0.5℃, it indicates that a mutation has occurred at the c.126G>C mutation site of the POLH gene.

[0037] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 63.2±0.5℃, it indicates that no mutation has occurred at the c.332G>A mutation site of the POLH gene.

[0038] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 61.1±0.5℃, it indicates that a mutation has occurred at the c.332G>A mutation site of the POLH gene.

[0039] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 81.2±0.5℃, it indicates that no mutation has occurred at the c.364A>C mutation site of the POLH gene.

[0040] When the melting curve temperature of the FAM fluorescence channel reaches a peak at 78.8±0.5℃, it indicates that a mutation has occurred at the c.364A>C mutation site of the POLH gene.

[0041] When the melting curve temperature of the VIC fluorescence channel reaches a peak at 65.2±0.5℃, it indicates that no mutation has occurred at the c.490G>T mutation site of the POLH gene.

[0042] When the melting curve temperature of the VIC fluorescence channel reaches a peak at 63.1±0.5℃, it indicates that a mutation has occurred at the c.490G>T mutation site of the POLH gene.

[0043] When the melting curve temperature of the VIC fluorescence channel peaks at 74.3±0.5℃, it indicates that no mutation has occurred at the c.1066C>T mutation site of the POLH gene.

[0044] When the melting curve temperature of the VIC fluorescence channel reaches a peak at 72.1±0.5℃, it indicates that a mutation has occurred at the c.1066C>T mutation site of the POLH gene.

[0045] When the melting curve temperature of the VIC fluorescence channel peaks at 69.8±0.5℃, it indicates that no mutation has occurred at the c.437dup T mutation site of the POLH gene.

[0046] When the melting curve temperature of the VIC fluorescence channel reaches a peak at 67.5±0.5℃, it indicates that a mutation has occurred at the c.437dup mutation site of the POLH gene.

[0047] As one of the objectives of this invention, this invention also provides a diagnostic kit, comprising at least an XPV melting curve mutation point detection solution, a wild-type positive control, a mutant positive control, and a negative control; the XPV melting curve mutation point detection solution comprises a probe-primer mixture, a PCR reaction solution, and an enzyme solution; the probe-primer mixture contains probes and primers with sequences such as SEQ.ID NO.1-11; the PCR reaction solution and enzyme solution are Novizan ChamQ Geno-SNP Probe Master MixQ811-02.

[0048] As one of the objectives of the invention, the present invention also provides a method for detecting XPV / POLH single base mutations, or the application of the kit described above in XPV / POLH gene detection for non-disease diagnosis purposes.

[0049] The beneficial effects of the technical solution provided by this invention compared with the prior art are as follows:

[0050] 1. This invention provides a method for detecting single-base mutations in the XPV / POLH gene, which is fast, convenient, and extremely inexpensive, and can be completed without complex equipment.

[0051] 2. Based on nucleic acid extraction methods, this invention establishes a method for detecting missense and nonsense mutations in the POLH gene using melting curves. This method is simple to operate, requires only basic PCR knowledge, and is quick to complete, taking only about 2-3 hours. No special equipment is needed; conventional quantitative PCR instruments can be used to interpret the results. The detection method is simple, low-cost, comparable to real-time quantitative PCR, and can provide a rapid, reliable, and accurate basis for the auxiliary diagnosis of XPV.

[0052] 3. By adopting the technical solution of the present invention, nucleic acid extraction from peripheral blood of XPV does not require special instruments; a pipette is sufficient. The extraction operation is simple and simplifies complex operations. In particular, the required material cost is low, which greatly reduces the cost of detection.

[0053] 4. The technical solution of this invention does not require special expensive instruments to detect mutation points by drawing melting curves. A conventional real-time fluorescence PCR instrument is sufficient. It is simple to operate and does not require professional personnel. The relevant testing personnel can complete the test by following the instructions. The detection time, from sampling to the issuance of interpretation results, can be completed within 2 hours, which greatly shortens the time for batch testing. It is suitable for hospitals and other relevant units to promote and use.

[0054] 5. The XPV melting curve detection mutation point coverage provided by this invention accounts for 45.2% of known mutation points. It can not only detect nonsense or missense mutations in general, providing information for subsequent clinical phenotypes, but also detect specific mutation sites based on specific nonsense or missense mutations. Attached Figure Description

[0055] Figure 1 This is the detection result of the mutation site c.25G>T in a typical embodiment of the present invention.

[0056] Figure 2 This is the detection result of the mutation site c.126G>C in a typical embodiment of the present invention.

[0057] Figure 3 This is the detection result of the mutation site c.332G>A in a typical embodiment of the present invention.

[0058] Figure 4 This is the detection result of the mutation site c.364A>C in a typical embodiment of the present invention.

[0059] Figure 5 This is the detection result of the mutation site c.490G>T in a typical embodiment of the present invention.

[0060] Figure 6 This is the detection result of the mutation site c.1066C>T in a typical embodiment of the present invention.

[0061] Figure 7 This is the detection result of the mutation site c.437dup in a typical embodiment of the present invention. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0063] Studies have shown that in the phenotypic-genotypic relationship of xeroderma pigmentosum (XPV), POLH gene mutations and mutation types are associated with clinical severity. There are 84 pathogenic POLH gene mutations associated with XPV, including missense mutations, nonsense mutations, splicing mutations, and insertion / deletion mutations. Nonsense and missense mutations account for 45.2% of these. Specifically, mutation information for XPV (POLH) is shown in Table 1. Missense mutation sites include c.25G>T, c.126G>C, c.332G>A, and c.364A>C, while nonsense mutation sites include c.490G>T, c.1066C>T, and c.437dup.

[0064] Table 1. Information on XPV (POLH) mutation points

[0065] Serial Number mutation site Mutation type Influence 1 c.25G>T Missense mutation Located in the N-terminal domain, it may interfere with protein localization. 2 c.126G>C Missense mutation Affecting the DNA binding interface, leading to loss of function. 3 c.332G>A Missense mutation It affects DNA binding ability, leading to reduced enzyme activity. 4 c.364A>C Missense mutation Located in a conserved region, it may affect enzyme stability. 5 c.490G>T nonsense mutation Premature termination of translation resulted in truncated protein. 6 c.1066C>T nonsense mutation Truncation mutations affect enzyme integrity. 7 c.437dup nonsense mutation Frameshift mutations causing truncation are common in XPV patients.

[0066] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0067] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The PCR reagents used in this invention are all from Nanjing Novizan Biotechnology Co., Ltd.

[0068] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0069] Example 1

[0070] This embodiment provides a method for detecting single-base mutations in the XPV / POLH gene, the specific steps of which include:

[0071] 1. Rapid extraction of nucleic acids from peripheral blood genome

[0072] 1) Select an EDTA anticoagulant tube and collect 2 mL of venous blood.

[0073] 2) Centrifuge the blood collection tube at 3000 rpm for 5 minutes. At this time, the suspension in the test tube can be seen to be divided into 3 layers: the upper layer is pale yellow plasma, the bottom layer is red blood cells, and there is a grayish-white white blood cell layer (normal human peripheral blood white blood cells) closely attached to the red blood cell layer.

[0074] 3) Use a capillary tube to draw up the cell suspension rich in white blood cells located on the red blood cell layer and transfer it to another test tube.

[0075] 4) Preparation of leukocyte lysis buffer:

[0076] ① Preparation of 1M Tris-HCl pH7.5: Weigh 121.1g of Tris and place it in a 1L beaker. Add about 800mL of deionized water, stir thoroughly to dissolve, and add concentrated hydrochloric acid to adjust the pH value to 7.5.

[0077] ② Add 50 mL of 1M Tris-HCl (pH 7.5), 15 g Tritonx-100, 10 g Sodium deoxycholate, 2 g SDS (sodium dodecyl sulfate), and 4 μL of 20 mg / mL proteinase K to 800 mL of deionized water, and then bring the volume to 1 L with deionized water.

[0078] 5) Take 300 μL of leukocyte lysis buffer, add 30 μL of leukocyte solution, and then vortex to mix for 1 min to obtain a mixture. This mixture is used as a nucleic acid sample for subsequent testing.

[0079] 2. Primer and probe design

[0080] Using XPV (POLH) sequence information and nonsense and missense mutation site information of the POLH gene, missense and nonsense mutation primers and probes were designed using Primer Express 3.0.1 software; the primers and probes were purified by HPLC.

[0081] Primer and probe sequences are shown in Table 2.

[0082] Table 2. Primer and probe sequences

[0083]

[0084] Note: + indicates that the following base is modified with locked nucleic acid.

[0085] 3. Preparation of primer and probe mixture

[0086] The preparation method includes: dissolving the synthesized primers and probes to 100 μM using TE buffer, and then preparing a primer-probe mixture. The preparation volume is shown in Table 3.

[0087] Table 3 Primer and probe ratios

[0088] Serial Number Raw material concentration 1 reaction volume / μL Reaction concentration / nM SEQ.ID NO.1 100μM 0.025 50 SEQ.ID NO.2 100μM 0.25 500 SEQ.ID NO.3 100μM 0.025 50 SEQ.ID NO.4 100μM 0.25 500 SEQ.ID NO.5 100μM 0.25 500 SEQ.ID NO.6 100μM 0.225 450 SEQ.ID NO.7 100μM 0.26 520 SEQ.ID NO.8 100μM 0.29 580 SEQ.ID NO.9 100μM 0.25 500 SEQ.ID NO.10 100μM 0.3 600 SEQ.ID NO.11 100μM 0.29 580 TE buffer / 12.585 / Total volume / 15 /

[0089] 4. Melting curve PCR reaction

[0090] This embodiment also includes negative and positive controls. The positive control is a synthetic plasmid, including a wild-type POLH plasmid and a mutant POLH plasmid, the sequences of which are shown in Table 4. The concentration of the wild-type POLH plasmid in the wild-type positive control and the concentration of the mutant POLH plasmid in the mutant positive control are both 10. 6 ±5000 copies / mL.

[0091] Table 4

[0092]

[0093]

[0094] The negative control was sterile water for injection.

[0095] 5. Preparation of the XPV melting curve abrupt change point detection solution

[0096] Take 15 μL of primer-probe mixture, 25 μL of ChamQ Geno-SNP Probe Master Mix (supplier: Novizan; catalog number: Q811-02) and 10 μL of nucleic acid sample or positive control / negative control extracted in step 1, and mix them to obtain XPV melting curve mutation point detection solution. In this example, Novizan's ChamQ Geno-SNP Probe Master Mix (catalog number: Q811-02) includes RT-PCR reaction solution and enzyme mixture.

[0097] 6. PCR reaction

[0098] The XPV melting curve mutation point detection solution was aliquoted into PCR reaction tubes at a volume of 40 μL, and the reaction was run using a real-time quantitative PCR instrument. The real-time quantitative PCR instrument was run according to the reaction program, which included: 95℃×2 min; 95℃×10 min, 60℃×1 min (40 cycles); 95℃×10 s, 55~85℃ (0.03℃ / s); the FAM and VIC fluorescence channels were set at 60℃×1 min and 60~90℃ for illumination.

[0099] 7. Result Interpretation

[0100] After the experiment, the Tm values ​​of the melting curve peaks were analyzed to interpret the results. For detailed interpretation, please refer to [link to relevant documentation]. Figures 1-7 See Table 5.

[0101] Table 5 Interpretation of Test Results

[0102] aisle Mutation point Mutation type Tm value of wild-type melting curve Tm value of abrupt melting curve FAM c.25G>T Missense mutation Tm = 65 ± 0.5℃ Tm=63±0.5℃ FAM c.126G>C Missense mutation Tm=69±0.5℃ Tm = 67 ± 0.5℃ FAM c.332G>A Missense mutation Tm=75±0.5℃ Tm=72±0.5℃ FAM c.364A>C Missense mutation Tm=81±0.5℃ Tm=79±0.5℃ VIC c.490G>T nonsense mutation Tm=72±0.5℃ Tm=69±0.5℃ VIC c.1066C>T nonsense mutation Tm = 74.3 ± 0.5℃ Tm = 72.1 ± 0.5℃ VIC c.437dup nonsense mutation Tm = 69.8 ± 0.5℃ Tm = 67.5 ± 0.5℃

[0103] See Figure 1 The results show the detection of c.25G>T wild-type, mutant, and heterozygous types. The melting curve temperature of the FAM fluorescence channel peaked at 68.9±0.5℃, indicating that no mutation occurred at the c.25G>T mutation site of the POLH gene. The melting curve temperature of the FAM fluorescence channel peaked at 66.6±0.5℃, indicating that a mutation occurred at the c.25G>T mutation site of the POLH gene.

[0104] See Figure 2The results show the detection of c.126G>C wild-type, mutant, and heterozygous types. The melting curve temperature of the FAM fluorescence channel peaked at 75.1±0.5℃, indicating that no mutation occurred at the c.126G>C mutation site of the POLH gene. The melting curve temperature of the FAM fluorescence channel peaked at 72.8±0.5℃, indicating that a mutation occurred at the c.126G>C mutation site of the POLH gene.

[0105] See Figure 3 The results show the detection results for wild-type, mutant, and heterozygous c.332G>A. The melting curve temperature of the FAM fluorescence channel shows a peak at 63.2±0.5℃, indicating that no mutation has occurred at the c.332G>A mutation site of the POLH gene. The melting curve temperature of the FAM fluorescence channel shows a peak at 61.1±0.5℃, indicating that a mutation has occurred at the c.332G>A mutation site of the POLH gene.

[0106] See Figure 4 The results show the detection of wild-type, mutant, and heterozygous c.364A>C. A peak temperature of 81.2±0.5℃ in the melting curve of the FAM fluorescence channel indicates that no mutation has occurred at the c.364A>C mutation site of the POLH gene. When a peak temperature of 78.8±0.5℃ in the melting curve of the FAM fluorescence channel indicates that a mutation has occurred at the c.364A>C mutation site of the POLH gene.

[0107] See Figure 5 The results show the detection of c.490G>T wild-type, mutant, and heterozygous types. The melting curve temperature of the VIC fluorescence channel peaked at 65.2±0.5℃, indicating that no mutation occurred at the c.490G>T mutation site of the POLH gene. The melting curve temperature of the VIC fluorescence channel peaked at 63.1±0.5℃, indicating that a mutation occurred at the c.490G>T mutation site of the POLH gene.

[0108] See Figure 6 The results show the detection of c.1066C>T wild-type, mutant, and heterozygous types. The melting curve temperature of the VIC fluorescence channel peaked at 74.3±0.5℃, indicating that no mutation occurred at the c.1066C>T mutation site of the POLH gene. The melting curve temperature of the VIC fluorescence channel peaked at 72.1±0.5℃, indicating that a mutation occurred at the c.1066C>T mutation site of the POLH gene.

[0109] See Figure 7The results show the detection of wild-type, mutant, and heterozygous c.437dup. The melting curve temperature of the VIC fluorescence channel peaked at 69.8±0.5℃, indicating that no mutation occurred at the c.437dup T mutation site of the POLH gene. The melting curve temperature of the VIC fluorescence channel peaked at 67.5±0.5℃, indicating that a mutation occurred at the c.437dup mutation site of the POLH gene.

[0110] Based on the above detection of nonsense and missense mutation sites in the POLH gene, any single base mutation site in the POLH gene can be detected using the method of this invention. That is, this invention determines whether there is an XPV / POLH genotype mutation by interpreting the detection sites of the XPV / POLH gene. In particular, using the technical solution of this invention, the false detection, misdetection, and missed detection rates are extremely low. The method of this invention can be used to detect other types of single base mutations in xeroderma pigmentosum, thereby making a comprehensive judgment on the disease and thus better preventing XP disease.

[0111] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A method for detecting XPV / POLH single-base mutations, characterized in that, Using the nucleic acid of the sample to be tested as template DNA, real-time fluorescence acquisition is performed through PCR reaction, amplification curve and melting curve are plotted, and the results are interpreted. The interpretation of the results includes the interpretation of the mutation sites of four missense mutations and three nonsense mutations in the POLH gene.

2. The method for detecting XPV / POLH single-base mutations according to claim 1, characterized in that, In the PCR reaction, the PCR amplification reaction system includes nucleic acid samples, XPV melting curve mutation point detection solution, positive control, and negative control; The XPV melting curve mutation point detection solution includes a primer and probe composition, a PCR reaction solution, and an enzyme solution. The amplified sequence of the wild-type plasmid is shown in SEQ ID NO.12; The amplified sequence of the mutant plasmid is shown in SEQ ID NO.13; The negative control was sterile water for injection.

3. The method for detecting XPV / POLH single-base mutations according to claim 2, characterized in that, The primer-probe composition includes a primer-probe combination for detecting non-mutated primers and a primer-probe combination for detecting mutations; In the detection primer-probe combination, at least one artificial mutation site is introduced on the adjacent base at the 5' end of the mutation site detected and analyzed by the probe, and the base of the artificial mutation site is modified with locked nucleic acid. The primer and probe compositions were purified by HPLC. The primer-probe combination for detecting non-mutated POLH genes includes primers and probes for detecting non-mutated POLH genes. The mutation detection primer-probe combination includes primers and probes for detecting mutations in the POLH gene.

4. The method for detecting XPV / POLH single-base mutations according to claim 3, characterized in that, In the unmutated primer-probe combination, the sequences of the primers and probes are shown in SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, and SEQ ID NO.: 4; In the detection mutation primer-probe combination, the sequences of the primers and probes are shown in SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, and SEQ ID NO.:

11.

5. The method for detecting XPV / POLH single-base mutations according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Nucleic acid extraction: After centrifugation of peripheral blood samples, the leukocyte fluid is collected, added to leukocyte lysis buffer for lysis, and the supernatant is removed to obtain nucleic acid samples for later use; S2. Reagent preparation: Take the probe primer mixture, PCR reaction solution, and enzyme solution, mix them evenly, and obtain the XPV melting curve mutation point detection solution; S3. Amplification and detection: Take nucleic acid sample, XPV melting curve mutation point detection solution, negative control, wild-type positive control and mutant positive control, mix them to obtain PCR amplification reaction system, perform melting curve PCR amplification reaction, and perform real-time fluorescence quantitative detection on a fluorescence quantitative PCR instrument; S4. Plot the PCR curve and melting curve, and interpret and analyze the test results.

6. The method for detecting XPV / POLH single-base mutations according to claim 5, characterized in that, In S1, the leukocyte lysis buffer consisted of 50 mM Tris-HCl pH 7.5, 150 mM KOH, 1 mM EDTA, 1.5% Tritonx-100, 1% Sodium deoxycholate, 0.2% SDS, and 80 μg / mL proteinase K, and the volume was adjusted to 100 μL with physiological saline. And / or, the cell lysis buffer and leukocyte fluid are mixed at a volume ratio of 100:1, and then vortexed for 1 min to mix. In S3, the wild-type positive control is a TE buffer solution containing a wild-type plasmid with the POLH gene; the mutant positive control is a TE buffer solution containing a mutant plasmid with the POLH gene, wherein the mutation refers to a missense mutation in the POLH gene, including mutation sites c.25G>T, c.126G>C, c.332G>A, c.364A>C, or a nonsense mutation including mutation sites c.490G>T, c.1066C>T, c.437dup. The concentration of wild-type plasmid in the wild-type positive control and the concentration of mutant plasmid in the mutant positive control were both 10. 6 ±5000 copies / mL.

7. The method for detecting XPV / POLH single-base mutations according to any one of claims 5, characterized in that, The amplification program for the PCR reaction was as follows: 95℃×2min; 95℃×10min, 60℃×1min (40 cycles); 95℃×10s, 55-85℃ (0.03℃ / s); FAM and VIC fluorescence channels were set at 60℃×1min and 60~90℃ for illumination.

8. The method for detecting XPV / POLH single-base mutations according to claim 5, characterized in that, Methods for interpreting test results include: When the melting curve temperature of the FAM fluorescence channel reaches a peak at 68.9±0.5℃, it indicates that no mutation has occurred at the c.25G>T mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 66.6±0.5℃, it indicates that a mutation has occurred at the c.25G>T mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 75.1±0.5℃, it indicates that no mutation has occurred at the c.126G>C mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 72.8±0.5℃, it indicates that a mutation has occurred at the c.126G>C mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 63.2±0.5℃, it indicates that no mutation has occurred at the c.332G>A mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 61.1±0.5℃, it indicates that a mutation has occurred at the c.332G>A mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 81.2±0.5℃, it indicates that no mutation has occurred at the c.364A>C mutation site of the POLH gene. When the melting curve temperature of the FAM fluorescence channel reaches a peak at 78.8±0.5℃, it indicates that a mutation has occurred at the c.364A>C mutation site of the POLH gene. When the melting curve temperature of the VIC fluorescence channel peaks at 65.2±0.5℃, it indicates that no mutation has occurred at the c.490G>T mutation site of the POLH gene. When the melting curve temperature of the VIC fluorescence channel reaches a peak at 63.1±0.5℃, it indicates that a mutation has occurred at the c.490G>T mutation site of the POLH gene. When the melting curve temperature of the VIC fluorescence channel reaches a peak at 74.3±0.5℃, it indicates that no mutation has occurred at the c.1066C>T mutation site of the POLH gene. When the melting curve temperature of the VIC fluorescence channel reaches a peak at 72.1±0.5℃, it indicates that a mutation has occurred at the c.1066C>T mutation site of the POLH gene. When the melting curve temperature of the VIC fluorescence channel peaks at 69.8±0.5℃, it indicates that no mutation has occurred at the c.437dupT mutation site of the POLH gene. When the melting curve temperature of the VIC fluorescence channel reaches a peak at 67.5±0.5℃, it indicates that a mutation has occurred at the c.437dup mutation site of the POLH gene.

9. A diagnostic reagent kit, characterized in that, It should include at least the XPV melting curve mutation point detection solution, wild-type positive control, mutant positive control, and negative control; The XPV melting curve mutation point detection solution includes probe primer mixture, PCR reaction solution, and enzyme solution; The probe-primer mixture contains probes and primers with sequences such as SEQ.ID NO.1 to 11; The PCR reaction solution and enzyme solution were Novozymes ChamQ Geno-SNP Probe Master MixQ811-02.

10. A method for detecting XPV / POLH single base mutations as described in any one of claims 1-8, or the kit as described in claim 9, for use in XPV / POLH gene detection for non-disease diagnosis purposes.

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