Human SLC25A13 gene detection kit and application

Abstract

This invention discloses a human SLC25A13 gene detection kit and its application, including primer and probe sets for eight related sites: c.851_854deL4, c.1638_1660dup, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T, and c.1078C>T. The probe design for the c.1638_1660dup site employs a competitive anti-slip design, enabling the nucleic acid combination 2 at this site to effectively distinguish between heterozygous and homozygous mutations at the c.1638_1660dup site. The kit provides reliable genotyping results with 100% consistency with direct sequencing, exhibiting higher sensitivity than traditional sequencing methods. Furthermore, it is simple and rapid to operate, facilitating large-scale deployment.

Description

Technical Field

[0001] This invention relates to the field of genotyping detection technology, and in particular to a human SLC25A13 gene detection kit and its application. Background Technology

[0002] Citrin Deficiency (CD) is an autosomal recessive genetic disorder caused by a biallelic mutation in the SLC25A13 gene. Neonatal intrahepatic cholestasis caused by Citrin Deficiency (NICCD) is currently the most common pediatric CD phenotype.

[0003] Newborns with Citrin deficiency are unable to metabolize many components of breast milk. Galactosemia resulting from breastfeeding is highly toxic to the eyes and brain and can cause severe steatorrhea, necessitating discontinuation of breastfeeding and switching to specialized formula. Otherwise, serious complications such as jaundice and cirrhosis may occur. Early diagnosis of NICCD patients and guidance on dietary adjustments can effectively prevent disease progression and reduce treatment risks and mortality. Therefore, conducting newborn screening for Citrin deficiency and disseminating knowledge about it in areas with a high incidence of this genetic disorder plays a crucial role in disease control and promoting healthy births.

[0004] The symptoms of NICCD are transient and complex, making it difficult to establish clear clinical diagnostic indicators. Currently, there are no universally recognized clinical or biochemical diagnostic criteria for NICCD at home and abroad. SLC25A13 gene mutation analysis is considered a reliable means of confirming NICCD. The presence of pathogenic mutations in both SLC25A13 alleles of a suspected patient is sufficient to confirm the disease.

[0005] Current research both domestically and internationally indicates that traditional analytical methods such as PCR-RFLP, LA-PCR DNA, and Sanger sequencing have limitations, including high sample requirements, complex equipment, long processing times, high costs, and difficulty in large-scale implementation. Chinese patent document CN103421909A addresses these issues by employing fluorescent PCR for SLC25A13 gene-related site mutation detection. Its innovation lies in its simple operation and low cost; however, it suffers from the risk of false negatives and false positives in result interpretation. Chinese patent document CN108048553A utilizes the more traditional Sanger sequencing method for SLC25A13 gene-related site mutation detection. This type of method is innovative in its ability to detect multiple sites, but its high cost is a drawback. Chinese patent document CN108753952A upgrades CN108048553A by employing multiplex PCR-sequencing, which allows for the detection of multiple SLC25A13 gene-related sites in a single PCR and sequencing process, reducing costs. However, it still suffers from operational complexity and time consumption. Next-generation sequencing, developed based on Sanger sequencing, has also been applied to the detection of mutations at SLC25A13 gene-related sites, as illustrated in Chinese patent document CN108913761A. Its innovation lies in its ability to detect a greater number of sites, but its drawbacks remain its complex operation, time consumption, and low degree of domestic instrument compatibility. Chinese patent document CN111073959A has successfully screened for mutations in several genes involved in Citrin deficiency using the fluorescence PCR melting curve method. This method overcomes the shortcomings of the aforementioned patents in terms of cost and ease of operation, but it increases the requirements for sample quality and carries the risk of melting curve drift. Furthermore, due to the limited number of sites detected, its clinical sensitivity and specificity are slightly lower.

[0006] Therefore, it is necessary to design a kit to enable convenient, time-saving, and reliable detection of genotypes at relevant loci, thereby improving detection sensitivity and specificity. Summary of the Invention

[0007] The purpose of this invention is to provide a human SLC25A13 gene detection kit to at least address one of the many shortcomings of the existing technology.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A human SLC25A13 gene detection kit includes primers and probes involved in the c.1638_1660dup site, wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 3-4, and the nucleotide sequences of the probes are shown in SEQ ID NO: 24-25.

[0010] In this invention, the aforementioned human SLC25A13 gene detection kit includes primer sets and probe sets related to the sites c.851_854deL4, c.1638_1660dup, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T, and c.1078C>T. The nucleotide sequences of the primer sets are shown in SEQ ID NO: 1-18; the nucleotide sequences of the probe sets are shown in SEQ ID NO: 22-37.

[0011] Furthermore, the kit also includes an endogenous internal standard with the nucleotide sequence shown in SEQ ID NO: 19-21.

[0012] Furthermore, the 5' end of the probe is modified with a fluorescent reporter group, and the 3' end is modified with a fluorescent quencher group.

[0013] Preferably, the fluorescent reporter group includes one or more of FAM, VIC, ROX, NED, Texred, CY3, and CY5; the fluorescent quenching group modified at the 3' end of the specific nucleic acid sequences SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 25 includes one or more of TAMARA, BHQ1, and BHQ2; and the fluorescent quenching group modified at the 3' end of the specific nucleic acid sequences SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 26 to SEQ ID NO: 37 is MGB.

[0014] Preferably, the working concentration of the nucleic acid sequence SEQ ID NO: 1~37 is 50~600 nmol / L.

[0015] The invention also includes a Taq enzyme, an UNG enzyme, a positive control, and a negative control in the aforementioned human SLC25A13 gene detection kit.

[0016] The present invention also provides the application of the above-described human SLC25A13 gene detection kit in the preparation of products for detecting human genomic DNA, wherein the human genomic DNA is derived from one or more of human blood, blood spots, saliva, saliva spots, and amniotic fluid.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] In the probe design process for the c.1638_1660dup site, this invention employs a competitive anti-slip design, enabling nucleic acid combination 2 at this site to effectively distinguish between heterozygous and homozygous mutations at the c.1638_1660dup site. Furthermore, given the difficulty in specifically distinguishing between heterozygous and homozygous mutations at the c.1638_1660dup site using the Taq-man fluorescent probe method, this invention adds the c.1638_1660dup site (which is one of the four major prevalent mutation sites in China, accounting for approximately 9% of patients), thereby improving the detection sensitivity of this invention.

[0019] The Taq-man fluorescent probe method of this invention has higher sensitivity than methods such as first-generation sequencing and reverse dot blot hybridization. After adopting the Taq-man fluorescent probe method and competitive anti-slip design method, the detection limit of the reagents or kits of this invention is as low as 0.4 ng / μL, which can further improve the detection sensitivity of this invention.

[0020] The detection kit provided by this invention provides reliable results for human SLC25A13 genotyping, with a 100% consistency rate with direct sequencing. It also has higher sensitivity than traditional sequencing methods and is simple and fast to operate, which is conducive to large-scale promotion. Attached Figure Description

[0021] Figure 1 This is the result of heterozygosity detection at the c.851_854deL4 site of the SLC25A13 gene in one clinical sample.

[0022] Figure 2 This is the result of wild-type detection of the c.1638_1660dup site of the SLC25A13 gene in one clinical sample.

[0023] Figure 3 This is the result of wild-type detection of the IVS6+5G>A site in the SLC25A13 gene in one clinical sample.

[0024] Figure 4 This is the result of wild-type detection of the IVS16ins3kb site in the SLC25A13 gene in one clinical sample.

[0025] Figure 5 This is the result of wild-type detection of the IVS4ins6kb site in the SLC25A13 gene in one clinical sample.

[0026] Figure 6 This is the result of wild-type detection of the IVS11+1G>A site in the SLC25A13 gene of a clinical sample.

[0027] Figure 7 This is the result of wild-type detection of the SLC25A13 gene c.1399C>T site in one clinical sample.

[0028] Figure 8 This is the result of wild-type detection of the SLC25A13 gene c.1078C>T site in one clinical sample.

[0029] Figure 9 The results of detecting the wild-type plasmid at the c.1638_1660dup site of the SLC25A13 gene using the probe in Comparative Scheme 1 are shown.

[0030] Figure 10 The results of detecting the mutant plasmid at the c.1638_1660dup site of the SLC25A13 gene using the probe in Comparative Scheme 1 are shown.

[0031] Figure 11 The results of detecting the wild-type plasmid at the c.1638_1660dup site of the SLC25A13 gene using the probe in Comparative Scheme 2 are shown.

[0032] Figure 12 The results of detecting the mutant plasmid at the c.1638_1660dup site of the SLC25A13 gene using the probe in Comparative Scheme 2 are shown. Detailed Implementation

[0033] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0034] In view of the detectable and treatable nature of citrulline deficiency and its serious clinical consequences, this invention provides a PCR amplification reagent or kit for simultaneously performing genotyping detection on eight relevant loci on the human SLC25A13 gene. The eight relevant loci on the SLC25A13 gene are: c.851_854deL4, c.1638_1660dup, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T, and c.1078C>T.

[0035] Under normal circumstances, the Taq-man fluorescent probe method has difficulty specifically distinguishing between heterozygous and homozygous mutations at the c.1638_1660dup site. Therefore, currently published patents using the Taq-man fluorescent probe method have avoided this site (application publication number CN103421909 A) to prevent the reagent from being unable to distinguish between heterozygous and homozygous mutations at the c.1638_1660dup site. This site is one of the four major prevalent genotypes in China. Therefore, through inventive work, the inventors discovered that the probe sequence designed in this invention can clearly distinguish between heterozygous and homozygous mutations at the c.1638_1660dup site, thus improving the sensitivity of the reagent.

[0036] Conventional probe design suffers from the following problem: when a template has multiple repeating (n≥2) copy regions, and Taq-man probes are used to distinguish between different copy numbers in this region, slippage (bubbling) occurs during probe-template binding. This results in the formation of various similar secondary structures between the probe and template, making it impossible to distinguish templates with different copy numbers. Through inventive effort, the inventors discovered that a special competitive anti-slip design method can be used to design a Taq-man probe that meets the requirements for distinguishing 1 / 2 copy templates. Therefore, a competitive anti-slip Taq-man probe was designed at the c.1638_1660dup site.

[0037] On one hand, the present invention provides a PCR amplification reagent or kit for simultaneously performing genotyping detection of eight related sites on the human SLC25A13 gene, comprising: specific nucleic acid sequences SEQ ID NO: 1 to SEQ ID NO: 18, SEQ ID NO: 22 to SEQ ID NO: 37 for eight related sites, namely c.851_854deL4, c.1638_1660dup, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T and c.1078C>T, and endogenous internal standard specific nucleic acid sequences SEQ ID NO: 19 to SEQ ID NO: 21.

[0038] Optionally, in some embodiments, the fluorescent reporter group modified at the 5' end of the specific nucleic acid sequences SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36 is FAM; and the fluorescent quencher group modified at the 3' end of the specific nucleic acid sequences SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36 is BHQ1.

[0039] Optionally, in some embodiments, the fluorescent reporter group modified at the 5' end of the specific nucleic acid sequences SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 37 is VIC; and the fluorescent quencher group modified at the 3' end of the specific nucleic acid sequences SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 37 is BHQ1.

[0040] Optionally, in some embodiments, the fluorescent reporter group modified at the 5' end of the specific nucleic acid sequence SEQ ID NO: 21 is ROX; and the fluorescent quencher group modified at the 3' end of SEQ ID NO: 21 is BHQ2.

[0041] Optionally, in some embodiments, the working concentration of the specific nucleic acid sequences SEQ ID NO: 1 to SEQ ID NO: 20 is 50 to 600 nmol / L.

[0042] Optionally, in some embodiments, the working concentration of the specific nucleic acid sequences SEQ ID NO: 21 to SEQ ID NO: 37 is 50 to 600 nmol / L.

[0043] Combining primers and probes from different gene loci in a single PCR reaction system requires consideration of interference between different primers and probes to avoid non-specific amplification. Furthermore, patent CN 111073959 A only detects four relevant loci of the SLC25A13 gene, resulting in slightly lower sensitivity and specificity, and carries the risk of melting curve drift. Through the inventors' inventive efforts, it was discovered that using the following eight different multiplex PCR reaction systems is convenient, time-saving, avoids interference between primers and probes to prevent non-specific amplification, and improves detection efficiency.

[0044] Therefore, the present invention also provides a human SLC25A13 gene detection reagent or kit, characterized in that it comprises: eight different multiplex PCR reaction systems for detecting eight sites of the SLC25A13 gene;

[0045] The eight different multiplex PCR reaction systems include eight different nucleic acid combinations and PCR buffers containing magnesium ions, dNTPs, KCl, Tris, ammonium sulfate, etc.

[0046] The eight different nucleic acid combinations include the following:

[0047] Nucleic acid combination 1: It is used to detect c.851_854deL4, and includes the nucleic acids shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0048] Nucleic acid combination 2: for detecting c.1638_1660dup, comprising the nucleic acids shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0049] Nucleic acid combination 3: for detecting IVS6+5G>A, which includes the nucleic acids shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0050] Nucleic acid combination 4: for detecting IVS16ins3kb, comprising the nucleic acids shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0051] Nucleic acid combination 5: for detecting IVS4ins6kb, comprising the nucleic acids shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0052] Nucleic acid combination 6: for detecting IVS11+1G>A, which includes the nucleic acids shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0053] Nucleic acid combination 7: for detecting c.1399C>T, comprising the nucleic acids shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21;

[0054] Nucleic acid combination 8: for detecting c.1078C>T, comprising the nucleic acids shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

[0055] Optionally, in some embodiments, the SLC25A13 genotyping kit may further include components such as Taq enzyme, UNG enzyme, positive control, and negative control.

[0056] On the other hand, the present invention provides a method for typing human SLC25A13 gene-related loci, the method comprising:

[0057] (1) Extract human genomic DNA and quantify the concentration of genomic DNA using ultraviolet spectrophotometry;

[0058] (2) Add the extracted genomic DNA for multiplex fluorescent PCR amplification;

[0059] (3) Determine the genotype of the 8 relevant sites of the DNA to be tested based on the Ct value of the multiplex fluorescent PCR amplification reaction.

[0060] Optionally, in some embodiments, the human genomic DNA is derived from one or more of human blood, blood spots, saliva, saliva spots, and amniotic fluid.

[0061] The following are specific experimental embodiments of the present invention, which are only preferred embodiments of the present invention and do not represent a limitation on the specific solutions of the present invention. The selection of some materials and methods are optional for those skilled in the art, but alternative solutions to the overall coverage principle are all within the scope of protection of the present invention. Example 1

[0062] The preparation of the SLC25A13 genotyping detection kit of the present invention includes the following steps:

[0063] 1. Primer and probe synthesis:

[0064] Eight sets of specific primers (SEQ ID NO:1~SEQ ID NO:18) and eight sets of specific probes (SEQ ID NO:22~SEQ ID NO:37) were designed and synthesized. The 5' end of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:36 was labeled with a FAM fluorescent group, and the 3' end was labeled with an MGB fluorescence quencher (SEQ ID NO:24 labeled BHQ-1). The 5' end of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37 was labeled with a VIC fluorescent group, and the 3' end was labeled with an MGB fluorescence quencher (SEQ ID NO:25 labeled BHQ-1). Primers and probes were prepared into 100 μM stock solutions for storage. See Table 1 for details.

[0065] Table 1: Primer and probe sequences

[0066]

[0067]

[0068]

[0069] 2. Preparation of internal standard system

[0070] A pair of internal standard primers targeting the human genome were designed and synthesized. The sequences of the primer pair are SEQ ID NO: 19 and SEQ ID NO: 20. An internal standard probe, SEQ ID NO: 21, was designed and synthesized, with a ROX fluorescent group labeled at the 5' end and a BHQ-2 fluorescence quencher labeled at the 3' end. The internal standard primers and the internal standard probe were prepared into 100 μM stock solutions for storage.

[0071] 3. Preparation of other reagents

[0072] Prepare a PCR buffer containing 6.0 mM MgCl2, 2.0 mM each of dATP, dUTP, dCTP, and dGTP; prepare an enzyme mixture containing 2.5 × 10⁻⁶ Taq enzyme. 3 U / mL, UNG enzyme 0.5×10 3 U / mL.

[0073] 4. Prepare positive and blank controls.

[0074] The positive control contains eight plasmid DNAs, which contain eight mutant genotype plasmids, namely c.851_854deL4, c.1638_1660dup, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T, and c.1078C>T, as described in the kit of this invention. The selection and design of these plasmids are well known to those skilled in the art, and each plasmid is 3000 copies / μL. The blank control is Tris-HCl (10mM) buffer.

[0075] 5. Preparation of PCR reaction solution

[0076] Prepare PCR reaction solutions for eight different systems as shown in Tables 2 to 9.

[0077] Table 2: Reaction system 1 - Detection of wild-type (FAM) and mutant (VIC) c.851_854deL4 sites

[0078]

[0079] Table 3: Reaction system 2-detection of wild-type (FAM) and mutant (VIC) c.1638_1660dup sites

[0080]

[0081] Table 4. Reaction system 3: Detection of wild-type (FAM) and mutant (VIC) IVS6+5G>A sites

[0082]

[0083] Table 5: Reaction system 4 - Detection of wild-type (FAM) and mutant (VIC) IVS16ins3kb sites

[0084]

[0085] Table 6: Reaction system 5 - Detection of wild-type (FAM) and mutant (VIC) IVS4ins6kb sites

[0086]

[0087] Table 7: Reaction system 6-Detection of wild-type (FAM) and mutant (VIC) IVS11+1G>A sites

[0088]

[0089] Table 8: Reaction system 7 - Detection of wild-type (FAM) and mutant (VIC) c.1399C>T sites

[0090]

[0091] Table 9: 8-Detection of wild-type (FAM) and mutant (VIC) c.1078C>T sites in the reaction system

[0092]

[0093] 6. Assemble the reagent kit

[0094] The kit contains 8 tubes / wells of PCR reaction solution for genotyping detection of c.851_854deL4, c.1638_1660dup, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T, and c.1078C>T loci, respectively. Based on the usage of each component in the PCR reaction system, the usage of each component in the 12-person and 24-person kits was calculated, and the components in each tube of the kit were prepared and assembled. Example 2

[0095] The lowest detection limit of the human SLC25A13 genotyping kit prepared in Example 1 was evaluated as a positive quality control kit.

[0096] Jurkat cell line genomic DNA was used to accurately quantify positive control samples. Two dilutions (2 ng / μL and 0.4 ng / μL) of the positive control at a concentration of 10 ng / μL were used as mutant templates, and two dilutions (2 ng / μL and 0.4 ng / μL) of Jurkat cell line genomic DNA were used as wild-type templates. The limit of detection (LOD) was evaluated for eight sites in eight reaction systems, with a ≥95% detection rate after 20 replicates as the evaluation criterion. The results are shown in the table below:

[0097] Table 10: Evaluation Results of the Limit of Detection

[0098]

[0099] The results showed that the lowest detection limit for the eight reaction systems was at least 0.4 ng / μL. Example 3

[0100] The human SLC25A13 genotyping kit prepared in Example 1 was used to test the sample.

[0101] In this embodiment, 120 outpatient anticoagulated whole blood samples were randomly collected, and genomic DNA was extracted from them. The genotypes of the SLC25A13 gene at 8 loci in the test samples were detected using the human SLC25A13 genotyping kit obtained in Example 1.

[0102] 1. Genomic DNA extraction from blood samples

[0103] Using the blood genomic DNA extraction kit from Tiangen Biotech Co., Ltd., follow the instructions as follows:

[0104] To prepare the required solution: Add anhydrous ethanol to buffer GD and wash buffer PW, referring to the volume on the label on the bottle.

[0105] Take 200 μL of blood sample, add 4 μL of RNase A (100 mg / mL) solution, vortex for 15 seconds, and let stand at room temperature for 5 minutes. Add 20 μL of Proteinase K solution and mix thoroughly. Add 200 μL of buffer GB, mix thoroughly by inverting, and incubate at 56°C for 10 minutes, inverting several times during incubation, until the solution becomes clear. Add 200 μL of anhydrous ethanol and mix thoroughly by inverting; flocculent precipitate may appear at this point. Add the resulting solution and flocculent precipitate to a CB3 adsorption column (place the adsorption column in a collection tube), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the CB3 adsorption column in the collection tube. Add 500 μL of buffer GD to the CB3 adsorption column (please check to ensure that anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the CB3 adsorption column in the collection tube. Add 600 μL of wash buffer PW to the adsorption column CB3 (please check to ensure anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column CB3 into the collection tube. Centrifuge at 12000 rpm for 2 minutes, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any remaining wash buffer in the adsorption material. Transfer the adsorption column CB3 to a 1.5 mL centrifuge tube, suspend 60 μL of elution buffer TB in the center of the adsorption membrane, incubate at room temperature for 2–5 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the solution at the bottom of the centrifuge tube. Take 2 μL of the obtained solution and use a spectrophotometer to detect the DNA concentration and quality. Take 20 ng (sample DNA diluted to 10 ng / μL) and add 2 μL to the kit prepared in Example 1 for the next PCR reaction. Store the remaining genomic DNA solution at -20℃.

[0106] 2. Quantitative real-time PCR detection of gDNA in samples

[0107] Take 2 μL of the diluted DNA sample from step 1 and add it sequentially to PCR reaction systems 1-8 of the Example 1 kit (23 μL each), making the total volume of each of the eight reaction systems 25 μL. Place the kit in a real-time PCR instrument and perform the amplification reaction after setting the PCR reaction program as shown below:

[0108] 37℃ for 10 min; 95℃ for 5 min; 95℃ for 15 s, 60℃ for 60 s, 40 cycles; after each cycle, the fluorescence signals of FAM, VIC and ROX were collected.

[0109] 3. Analysis of Sample Detection Results: The detection results of 120 samples are as follows:

[0110] One c.851_854deL4 heterozygous sample was tested, and the results are as follows: Figures 1-8 As shown; the test results of the remaining 119 cases were all wild-type at 8 loci of the SLC25A13 gene.

[0111] The results of fluorescence PCR detection in the above 120 samples were consistent with the sequencing results. These results indicate that the detection kit provided by this invention is reliable for human SLC25A13 genotyping, achieving 100% consistency with direct sequencing. Furthermore, the detection method of this invention has higher sensitivity than traditional sequencing methods, is simple and rapid to operate, and is suitable for large-scale deployment.

[0112] Comparative Example

[0113] Primers were designed according to conventional primer design methods, and probes were designed and synthesized according to conventional fluorescent PCR probe design principles and the competitive anti-slip probe design principles summarized by the inventors.

[0114] Primers F1 and R1, specific to the c.1638_1660dup site, were designed and synthesized; specific probes P1, P2, P3, and P4 were designed and synthesized. P1 and P3 were labeled with a FAM fluorescent group at the 5' end and a BHQ1 fluorescent quencher at the 3' end; P2 and P4 were labeled with a VIC fluorescent group at the 5' end; P2 was labeled with an MGB fluorescent quencher at the 3' end; and P4 was labeled with a BHQ1 fluorescent quencher at the 3' end. The synthesized primers and probes were prepared into 10 μM working solutions. The primer and probe sequences are detailed in Table 11.

[0115] Table 11: Primer and probe design schemes and nucleotide sequences

[0116]

[0117] 1. Optimal Probe Design Scheme

[0118] Prepare the PCR reaction system as shown in Table 12 (the only difference between the two schemes is the probe design; all other components are the same).

[0119] Table 12: PCR reaction system formulation for preferred probes

[0120]

[0121] The detection results of wild-type and mutant plasmids of the SLC25A13 gene c.1638_1660dup site using the probe in Scheme 1 are as follows: Figure 9-10As shown in the figure. The detection results of wild-type and mutant plasmids of the SLC25A13 gene c.1638_1660dup site using the probe in scheme 2 are as follows. Figure 11-12 As shown.

[0122] The results showed that the two design schemes were significantly different: the mutant probe in scheme 1 could distinguish mutant templates, but the wild-type probe could not distinguish between wild-type and mutant templates at all; both the wild-type probe and the mutant probe in scheme 2 could specifically distinguish between wild-type and mutant templates.

[0123] In summary, this invention innovatively employs the following measures in the diagnostic process:

[0124] First, a competitive anti-slip design was employed in the probe design for the c.1638_1660dup site, enabling nucleic acid combination 2 at this site to effectively distinguish between heterozygous and homozygous mutations at the c.1638_1660dup site. Second, given the difficulty of specifically distinguishing between heterozygous and homozygous mutations at the c.1638_1660dup site using the Taq-man fluorescent probe method, the c.1638_1660dup site (one of the four major prevalent mutation sites in China, accounting for approximately 9% of patients) was added in this invention, improving the detection sensitivity of this invention. Finally, the sensitivity of the Taq-man fluorescent probe method is higher than that of first-generation sequencing and reverse dot blot hybridization. After adopting the Taq-man fluorescent probe method and the competitive anti-slip design method, the detection limit of the reagents or kits of this invention is as low as 0.4 ng / μL, further improving the detection sensitivity of this invention.

[0125] This invention is not limited to the description in the specification and embodiments, and thus other advantages and modifications can be readily realized by those skilled in the art. Therefore, without departing from the spirit and scope of the general concept defined by the claims and their equivalents, this invention is not limited to the specific details, representative solutions and examples described herein. sequence list <110> Wuhan Youzhiyou Medical Technology Co., Ltd. <120> A human SLC25A13 gene detection kit and its application <160> 43 <170> SIPOSequenceListing 1.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <400> 1 ttggtatatt tgttgcttgt gtttgtt 27 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <400> 2 agcaatccgt tcaatgtctg c 21 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 tgcctgcagc atctttagtg a 21 <210> 4 <211> twenty four <212> DNA <213> Artificial Sequence <400> 4 aagcagtcta tcactccgct gtaa 24 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <400> 5 tgtcttgact ccttttgtag aagaatg 27 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 gagttaaacc acttcattag ggcaa 25 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <400> 7 gtgttcttaa ctaactcttt ggtatcaggt 30 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 cttcctcctc ccttggcag 19 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <400> 9 agcaattcgt aagtcagcaa agttac 26 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <400> 10 acagatttta tcagaaagaa cttcatttac ttt 33 <210> 11 <211> twenty four <212> DNA <213> Artificial Sequence <400> 11 tgtagaccgg agctgttcct attc 24 <210> 12 <211> twenty three <212> DNA <213> Artificial Sequence <400> 12 gttgatgaat tgtggtctgt cca 23 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <400> 13 cctatcgatc ttgtaaaaac tcgaatg 27 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <400> 14 tcacctaaca ggtattgagc atgtg 25 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 tttgcaagtg gcaggagaaa 20 <210> 16 <211> twenty one <212> DNA <213> Artificial Sequence <400> 16 gcacgacaga cagagcactg a 21 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <400> 17 cctatcgatc ttgtaaaaac tcgaatg 27 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <400> 18 tcacctaaca ggtattgagc atgtg 25 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <400> 19 gggccactag gcgctc 16 <210> 20 <211> 15 <212> DNA <213> Artificial Sequence <400> 20 agccacccgc gaact 15 <210> twenty one <211> 16 <212> DNA <213> Artificial Sequence <400> twenty one ctccctccgc gcagcc 16 <210> twenty two <211> 19 <212> DNA <213> Artificial Sequence <400> twenty two cctacagacg tatgacctt 19 <210> twenty three <211> 17 <212> DNA <213> Artificial Sequence <400> twenty three ccctacagac gacctta 17 <210> twenty four <211> 17 <212> DNA <213> Artificial Sequence <400> twenty four tggctgcccg ggctggc 17 <210> 25 <211> twenty four <212> DNA <213> Artificial Sequence <400> 25 cccggggaga ttacaggtgg ctgc 24 <210> 26 <211> twenty two <212> DNA <213> Artificial Sequence <400> 26 tctagtagct gtaagttgta ac 22 <210> 27 <211> twenty two <212> DNA <213> Artificial Sequence <400> 27 tctagtagct gtaaattgta ac 22 <210> 28 <211> twenty three <212> DNA <213> Artificial Sequence <400> 28 tgtgatttct ccattttttt aaa 23 <210> 29 <211> 17 <212> DNA <213> Artificial Sequence <400> 29 cccgatttct ccatttt 17 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <400> 30 aatcaacctg tcttttct 18 <210> 31 <211> 15 <212> DNA <213> Artificial Sequence <400> 31 ccagtctcgt ctttt 15 <210> 32 <211> twenty one <212> DNA <213> Artificial Sequence <400> 32 tggactgtat agaggttagt g 21 <210> 33 <211> twenty one <212> DNA <213> Artificial Sequence <400> 33 tggactgtat agagattagt g 21 <210> 34 <211> 17 <212> DNA <213> Artificial Sequence <400> 34 cactggtcct cgagtca 17 <210> 35 <211> 17 <212> DNA <213> Artificial Sequence <400> 35 cactggtcct tgagtca 17 <210> 36 <211> 17 <212> DNA <213> Artificial Sequence <400> 36 cagaaccaac gatcaac 17 <210> 37 <211> 17 <212> DNA <213> Artificial Sequence <400> 37 cagaaccaat gatcaac 17 <210> 38 <211> twenty two <212> DNA <213> Artificial Sequence <400> 38 ggtatgcctg cagcatcttt ag 22 <210> 39 <211> 25 <212> DNA <213> Artificial Sequence <400> 39 gcagtctatc actccgctgt aagtg 25 <210> 40 <211> 25 <212> DNA <213> Artificial Sequence <400> 40 cgagattaca ggtgactgcc cgggc 25 <210> 41 <211> 13 <212> DNA <213> Artificial Sequence <400> 41 ccggggagat tac 13 <210> 42 <211> 17 <212> DNA <213> Artificial Sequence <400> 42 gtggctgccc gggctgg 17 <210> 43 <211> twenty three <212> DNA <213> Artificial Sequence <400> 43 ccggggagat tacaggtggc tgc 23

Claims

1. A human SLC25A13 gene detection kit, characterized in that, The primer and probe set includes the site c.1638_1660dup; wherein the nucleotide sequences of the primers are shown in SEQ ID NO: 3-4, and the nucleotide sequences of the probes are shown in SEQ ID NO: 24-25.

2. The human SLC25A13 gene detection kit according to claim 1, characterized in that, The kit comprises multiple tubes, each independently containing a c.1638_1660dup site and at least one of the following sites: c.851_854deL4, IVS6+5G>A, IVS16ins3kb, IVS4ins6kb, IVS11+1G>A, c.1399C>T, or c.1078C>T, to detect the primer and probe set involved.

3. The human SLC25A13 gene detection kit according to claim 2, characterized in that, The primer-probe set for detecting the c.851_854deL4 site includes primers with nucleotide sequences as shown in SEQ ID NO: 1-2 and probes as shown in SEQ ID NO: 22-23; The primer and probe set for detecting the IVS6+5G>A site includes primers with nucleotide sequences as shown in SEQ ID NO: 5-6 and probes as shown in SEQ ID NO: 26-27. The primer and probe set for detecting the IVS16ins3kb site includes primers with nucleotide sequences as shown in SEQ ID NO: 7-9 and probes as shown in SEQ ID NO: 28-29; The primer and probe set for detecting the IVS4ins6kb site includes primers with nucleotide sequences as shown in SEQ ID NO: 10-12 and probes as shown in SEQ ID NO: 30-31; The primer and probe set for detecting the IVS11+1G>A site includes primers with nucleotide sequences as shown in SEQ ID NO: 13-14 and probes as shown in SEQ ID NO: 32-33; The primer and probe set for detecting the c.1399C>T site includes primers with nucleotide sequences as shown in SEQ ID NO: 15-16 and probes as shown in SEQ ID NO: 34-35; The primer and probe set for detecting the c.1078C>T site includes primers with nucleotide sequences as shown in SEQ ID NO: 17-18 and probes as shown in SEQ ID NO: 36-37.

4. The human SLC25A13 gene detection kit according to claim 3, characterized in that, The primer-probe set for any of the detection sites also includes an endogenous internal standard with the nucleotide sequence shown in SEQ ID NO: 19-21.

5. The human SLC25A13 gene detection kit according to claim 4, characterized in that, The probe is modified with a fluorescent reporter group at its 5' end and a fluorescent quencher group at its 3' end.

6. The human SLC25A13 gene detection kit according to claim 5, characterized in that, The fluorescent reporter group includes one or more of FAM, VIC, ROX, NED, Texred, CY3, and CY5; the fluorescent quenching group modified at the 3' end of the sequences SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 25 is selected from one or more of TAMARA, BHQ1, and BHQ2; the fluorescent quenching group modified at the 3' end of the sequences SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 26 to SEQ ID NO: 37 is MGB.

7. The human SLC25A13 gene detection kit according to claim 4, characterized in that, The working concentration of the nucleotide sequences SEQ ID NO: 1~37 is 50~600 nmol / L.

8. The human SLC25A13 gene detection kit according to claim 1, characterized in that, It also includes Taq enzyme, UNG enzyme, positive control and negative control.

9. The use of the detection kit according to any one of claims 1-8 in the preparation of a detection product for the human SLC25A13 gene c.1638_1660dup site.

10. The application according to claim 9, characterized in that, The human genomic DNA is derived from one or more of human blood, blood spots, saliva, saliva spots, and amniotic fluid.

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