Primer group, chip, kit and method for detecting mutation of Hilteline deficiency disease-causing gene SLC25A13

Abstract

The invention discloses a primer group, a chip, a kit and a method for detecting mutation of a virulence gene SLC25A13 of Hilteline deficiency. The primer group comprises a primer group 1 and / or a primer group 2. According to the method for detecting mutation IVS16ins3kb and IVS4ins6kb of the Hilteline deficiency disease-causing gene SLC25A13, provided by one embodiment of the invention, two insertion variation types IVS16ins3kb and IVS4ins6kb of the SLC25A13 gene can be detected at the same time through one-time multiple LA-PCR reaction, and the method has the characteristics of short time consumption, low cost and high specificity; and the method is suitable for conventional clinical verification work and is easy to popularize.

Description

Technical Field

[0001] The present application relates to the field of gene detection technology, and in particular to a primer set, chip, kit and method for detecting mutations in the citrin deficiency-causing gene SLC25A13. Background Art

[0002] Citrin deficiency, caused by mutations in the SLC25A13 gene, is an autosomal recessive metabolic disorder encompassing adult-onset citrullinemia type II (CTLN2) and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). CTLN2, the most common and severe phenotype, is characterized by frequent episodes of hyperammonemia, hepatic steatosis, neuropsychiatric symptoms, and cerebral edema.

[0003] Epidemiological studies have shown that the disease is pan-ethnic, primarily concentrated in East Asian countries. Common SNVs (single nucleotide variants) and indels (insertion-deletions) include c.851-854delGTAT (I), c.1638_1660dup (III), and IVS6 + 5G > (X). Common insertion variants include IVS16ins3kb (XIX) and IVS4ins6kb.

[0004] Citrin deficiency has yet to establish established clinical and biochemical diagnostic criteria. SLC25A13 gene mutation analysis is considered essential for diagnosis. Analytical techniques for detecting long-range variants primarily include long-range amplification PCR (LA-PCR), multiplex fluorescence quantitative PCR, and next-generation sequencing (NGS). These techniques are associated with complex procedures, high costs, and long testing cycles.

[0005] LA-PCR uses primers designed to amplify long fragments of the variant region. PCR amplification is then performed, and the presence of a mutation is determined by electrophoresis analysis of the size of the amplified product. This technique requires high standards for primer design and amplification conditions, and typically only amplifies a single product.

[0006] Multiplex fluorescence quantitative PCR (QPCR) combines QPCR with multiplex PCR. It uses multiple primer pairs and fluorescently labeled probes in a single reaction system to achieve real-time quantitative detection of multiple target sequences. This technology requires specialized QPCR instruments and multiple fluorescent probes, resulting in high costs and strict requirements for primer and probe design and reaction conditions.

[0007] NGS sequencing detects variations such as insertions, deletions, and inversions in gene sequences by analyzing split reads in sequencing data. For the detection of the IVS16ins3kb and IVS4ins6kb variants in the SLC25A13 gene, this method can identify the location of the mutation and can also detect single-base mutations. This technology requires specialized bioinformatics analysis skills and specific software tools, has high data analysis requirements, is relatively expensive, and may have some uncertainty in detecting rare or novel mutation sites.

[0008] Therefore, there is an urgent need to develop new detection primers and detection methods in this field, so as to quickly, accurately and conveniently detect the two common insertion variants IVS16ins3kb (XIX) and IVS4ins6kb. Summary of the Invention

[0009] Based on this, at least a primer set, a chip, a kit and a method for detecting mutations in the citrin deficiency-causing gene SLC25A13 are provided.

[0010] In a first aspect of the present application, a primer set is provided for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency causative gene SLC25A13, the primer set comprising:

[0011] Primer set 1: upstream and downstream primers with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 for amplifying IVS16ins3kb; and / or,

[0012] Primer set 2: upstream and downstream primers with sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 for amplifying IVS4ins6kb.

[0013] In a second aspect of the present application, a chip for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency-causing gene SLC25A13 is provided, which comprises the primer set described in the first aspect.

[0014] In the third aspect of the present application, there is provided use of the primer set described in the first aspect or the chip described in the second aspect in preparing a kit for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency causative gene SLC25A13.

[0015] In a fourth aspect of the present application, a kit is provided, comprising the primer set described in the first aspect and / or the chip described in the second aspect.

[0016] In some embodiments, the kit further comprises one or more of a nucleic acid releasing reagent, a nucleic acid extracting reagent, a DNA polymerase, dNTPs, and a PCR buffer.

[0017] In some embodiments, the DNA polymerase comprises one or more of Phanta enzyme and LA Taq enzyme.

[0018] In some embodiments, the kit further comprises a positive quality control and / or a negative quality control.

[0019] In some embodiments, the negative control does not contain the gene SLC25A13 with mutations IVS16ins3kb and IVS4ins6kb.

[0020] In some embodiments, the positive control comprises the gene SLC25A13 with mutations IVS16ins3kb and IVS4ins6kb.

[0021] In a fifth aspect of the present application, a method for using the kit according to the fourth aspect is provided, comprising the following steps:

[0022] extracting nucleic acid from the sample to be tested and preparing a nucleic acid sample;

[0023] performing LA-PCR amplification on the nucleic acid sample; and,

[0024] Obtain and analyze results.

[0025] In some embodiments, the sample to be tested includes one or more of peripheral blood, tissue, saliva, oral swab, and dried blood spot.

[0026] In some embodiments, in each reaction system for LA-PCR amplification: the initial system concentration of each primer is independently 5 μM to 15 μM, the initial system concentration of the nucleic acid sample is 50 ng / μL to 150 ng / μL, and the initial system concentration of dNTP is 5 mM to 15 mM.

[0027] In some embodiments, the DNA polymerase in the kit is Phanta enzyme, and the amount of Phanta used is (0.5 μL~2 μL) / 50 μL reaction system.

[0028] In some embodiments, the procedure for performing LA-PCR amplification comprises:

[0029] Pre-denaturation, 95°C, 3 minutes;

[0030] Denaturation, 95°C, 15 seconds, annealing, 60°C, 15 seconds, extension, 72°C, 4 minutes, 35 cycles;

[0031] Extend thoroughly at 72°C for 4 minutes.

[0032] In some embodiments, the PCR amplification is long fragment amplification PCR.

[0033] The method provided in one embodiment of the present application for detecting mutations IVS16ins3kb and IVS4ins6kb of the citrin deficiency-causing gene SLC25A13 can simultaneously detect two types of insertion mutations IVS16ins3kb and IVS4ins6kb of the SLC25A13 gene through a single multiplex LA-PCR reaction, and has the characteristics of short time consumption, low cost, and high specificity; the operation is simple and efficient, suitable for routine clinical verification work, and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0035] Figure 1 Schematic diagram of primer design for IVS16ins3kb in one embodiment of the present application.

[0036] Figure 2 Schematic diagram of IVS4ins6kb primer design in one embodiment of the present application.

[0037] Figure 3 The electrophoresis patterns of 8 normal control samples of SLC25A13 gene IVS16ins3kb and IVS4ins6kb in one embodiment of the present application are shown.

[0038] Figure 4This is the electrophoresis pattern of 8 SLC25A13 gene IVS16ins3kb heterozygous mutation samples in one embodiment of the present application.

[0039] Figure 5 This is the electrophoresis pattern of 5 SLC25A13 gene IVS4ins6kb heterozygous mutation samples in one embodiment of the present application.

[0040] Figure 6 The electrophoresis patterns of three SLC25A13 gene IVS16ins3kb and IVS4ins6kb heterozygous mutation samples in one embodiment of the present application are shown. DETAILED DESCRIPTION

[0041] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0044] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0045] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0046] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0047] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0048] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0049] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0050] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0051] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc. serve only for the purpose of non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0052] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0053] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0054] This application innovatively utilizes multiplex LA-PCR technology to successfully design primers and construct a precise and efficient detection method. This technology involves designing primers at both ends of the target region, amplifying and enriching the target sequence through multiplex LA-PCR, and then analyzing the PCR products by agarose gel electrophoresis. The electrophoresis pattern directly determines whether the sample contains IVS16ins3kb or IVS4ins6kb mutations. This primer and detection method enables simultaneous detection of both IVS16ins3kb and IVS4ins6kb mutations in SLC25A13 through a single LA-PCR reaction, meeting the need for simultaneous detection of IVS16ins3kb and IVS4ins6kb insertion mutations in the citrin deficiency-causing gene SLC25A13.

[0055] This detection method is a new type of rapid genetic diagnostic method in the field of biotechnology. It does not require DNA sequencing and is simple, fast, accurate, low-cost, and has direct and reliable test results. It is significantly superior to existing methods in terms of detection time and cost. It can be completed using simple and common equipment and is suitable for rapid detection of IVS16ins3kb and / or IVS4ins6kb mutations in the SLC25A13 gene and large-scale population screening by medical and testing institutions across the country.

[0056] In a first aspect of the present application, a primer set is provided for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency causative gene SLC25A13, the primer set comprising:

[0057] Primer set 1: upstream and downstream primers with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 for amplifying IVS16ins3kb; and / or,

[0058] Primer set 2: upstream and downstream primers with sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 for amplifying IVS4ins6kb.

[0059] In some embodiments, the primer set only includes primer set 1, which is used to detect the mutation IVS16ins3kb of the citrin deficiency-causing gene SLC25A13.

[0060] In some embodiments, the primer set only includes primer set 2, which is used to detect the mutation IVS4ins6kb of the citrin deficiency-causing gene SLC25A13.

[0061] In some embodiments, the primer set comprises primer set 1 and primer set 2, which can simultaneously detect mutations IVS16ins3kb and IVS4ins6kb of the citrin deficiency-causing gene SLC25A13.

[0062] During their experiments, the inventors discovered that some primers exhibited poor amplification performance. For example, a pair of primers designed to amplify the 6kb region of IVS4ins was overly long due to the mutation site. Improper design could result in low amplification efficiency and compromised accuracy. The inventors creatively optimized the amplification experimental system and primer sequences to overcome this issue, achieving efficient and accurate amplification of specific fragments.

[0063] The primer set finally obtained can amplify efficiently - by optimizing the annealing temperature and extension time, the experimental time is shortened, the amplification efficiency is improved, and the detection rate of the sample is ensured; and it has high specificity - accurately amplifying the target mutation and reducing false positives.

[0064] In a second aspect of the present application, a chip for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency-causing gene SLC25A13 is provided, which comprises the primer set described in the first aspect.

[0065] In the third aspect of the present application, there is provided use of the primer set described in the first aspect or the chip described in the second aspect in preparing a kit for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency causative gene SLC25A13.

[0066] In a fourth aspect of the present application, a kit is provided, comprising the primer set described in the first aspect and / or the chip described in the second aspect.

[0067] In some embodiments, the kit further comprises one or more of a nucleic acid releasing reagent, a nucleic acid extracting reagent, a DNA polymerase, dNTPs, and a PCR buffer.

[0068] The nucleic acid extraction reagent may be from a DNA extraction kit, such as a conventional extraction kit in the art.

[0069] In some embodiments, the brand of the DNA extraction kit is Changchun Zhiang Biotechnology, and its product number is GO-BTCDs.

[0070] Illustratively, the kit includes: the primer set as described in the first aspect, a nucleic acid releasing reagent, a nucleic acid extraction reagent, a DNA polymerase, dNTPs, and a PCR buffer.

[0071] In some embodiments, the DNA polymerase comprises one or more of Phanta enzyme and LA Taq enzyme.

[0072] In some embodiments, the kit further comprises a positive quality control.

[0073] In some embodiments, the kit further comprises a negative control.

[0074] In some embodiments, the kit further comprises a positive quality control and a negative quality control.

[0075] In some embodiments, the negative control does not contain the gene SLC25A13 with mutations IVS16ins3kb and IVS4ins6kb.

[0076] In some embodiments, the positive control comprises the gene SLC25A13 with mutations IVS16ins3kb and IVS4ins6kb.

[0077] In a fifth aspect of the present application, a method for using the kit according to the fourth aspect is provided, comprising the following steps:

[0078] S100 extracting nucleic acid from the test sample and preparing a nucleic acid sample;

[0079] S200. performing LA-PCR amplification on the nucleic acid sample; and,

[0080] S300. Obtain and analyze results.

[0081] The application scenario of the method of use is, for example, disease diagnosis or non-disease diagnosis (such as disease screening).

[0082] In some embodiments, the application scenarios of the method of use are, for example, disease screening, providing fertility risk assessment for families, and providing a basis for eugenics and good parenting.

[0083] In some embodiments, the method is used to detect genetic variations in immediate family members to verify whether they conform to the rules of autosomal recessive inheritance, thereby helping to reduce the risk of birth of sick offspring.

[0084] In some embodiments, the method of use is used to screen for SLC25A13 hotspot mutations in newborns, identify NICCD risk early, and guide clinical management and prognosis improvement.

[0085] In some embodiments, in step S100, the sample to be tested includes one or more of peripheral blood, tissue, saliva, oral swab, and dried blood spot.

[0086] In some embodiments, in step S200, in each reaction system for LA-PCR amplification: the initial system concentration of each primer is independently 5 μM to 15 μM, the initial system concentration of the nucleic acid sample is 50 ng / μL to 150 ng / μL, and the initial system concentration of dNTP is 5 mM to 15 mM.

[0087] Illustratively, in step S200, in each reaction system for LA-PCR amplification: the initial system concentration of each primer is independently 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, or a range or value between any two values.

[0088] Illustratively, in step S200, in each reaction system for LA-PCR amplification: the initial system concentration of the nucleic acid sample is 50 ng / μL, 60 ng / μL, 70 ng / μL, 80 ng / μL, 90 ng / μL, 100 ng / μL, 110 ng / μL, 120 ng / μL, 130 ng / μL, 140 ng / μL, 150 ng / μL, or a range or value between any two values.

[0089] Illustratively, in step S200, in each reaction system for LA-PCR amplification, the DNA polymerase is Phanta enzyme, and the amount of Phanta used is (0.5 μL-2 μL) / 50 μL reaction system. Illustratively, it is 0.5μL / 50μL, 0.6μL / 50μL, 0.7μL / 50μL, 0.8μL / 50μL, 0.9μL / 50μL, 1μL / 50μL, 1.1μL / 50μL, 1.2μL / 50μL, 1.3μL / 50μL, 1.4μL / 50μL, 1.5μL / 50μL, 1.6μL / 50μL, 1.7μL / 50μL, 1.8μL / 50μL, 1.9μL / 50μL, 2μL / 50μL, or a range or value between any two values.

[0090] Illustratively, in step S200, in each reaction system for LA-PCR amplification, the initial system concentration of dNTP is 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, or a range or value between any two values.

[0091] In some embodiments, the procedure for performing LA-PCR amplification comprises:

[0092] Pre-denaturation, 95°C, 3 minutes;

[0093] Denaturation, 95°C, 15 seconds, annealing, 60°C, 15 seconds, extension, 72°C, 4 minutes, 35 cycles;

[0094] Extend thoroughly at 72°C for 4 minutes.

[0095] In some embodiments, the LA-PCR amplification is long fragment amplification PCR.

[0096] In some embodiments, the method of use comprises:

[0097] 1) Extraction of sample genomic DNA

[0098] For samples such as peripheral blood, tissue, saliva, oral swabs, and dried blood spots, genomic DNA is extracted using nucleic acid extraction or purification reagents, and the extracted DNA is quality tested to obtain genomic DNA at a concentration of 50 ng / μL to 150 ng / μL.

[0099] 2) Multiplex LA-PCR amplification:

[0100] LA-PCR amplification was performed, and the primers used were shown in Table 1.

[0101] The relative amounts of different primers can be conventional relative amounts in the art, for example, the final concentration ratio of F1, R1, F2 and R2 can be 1:1:1:1.

[0102] The PCR amplification enzyme may be an enzyme conventionally used in the art for LA-PCR amplification, such as, for example, Phanta enzyme.

[0103] In some embodiments, the DNA amplification enzyme is a DNA amplification enzyme purchased from Vazyme with product number P505.

[0104] The PCR reaction system can be illustratively 50 μL. In some embodiments, the reaction system used can be referred to, for example, Table 1.

[0105] The LA-PCR reaction conditions are shown in Table 2.

[0106] 3) Agarose gel electrophoresis:

[0107] After the LA-PCR amplification product is electrophoresed in a conventional agarose gel (recommended 1%, 140V, 30 minutes), the DNA bands are observed using a gel imaging system.

[0108] 4) Interpretation of results and standards:

[0109] (1) Samples negative for both IVS16ins3kb and IVS4ins6kb: A 334 bp band was detected in F1 / R1 and a 1437 bp band was detected in F2 / R2.

[0110] (2) Heterozygous samples of IVS16ins3kb mutation: F1 / R1 detected 334bp and 3541bp bands, F2 / R2 detected 1437bp band; homozygous F1 / R1 detected 3541bp band; F2 / R2 detected 1437bp band.

[0111] (3) Heterozygous samples of the IVS4ins6kb mutation: a 334bp band was detected in F1 / R1; 1437bp and 7494bp bands were detected in F2 / R2. Homozygous F1 / R1 detected a 334bp band; F2 / R2 detected a 7494bp band.

[0112] (4) Samples containing both IVS16ins3kb and IVS4ins6kb mutations:

[0113] 1) When heterozygous for IVS16ins3kb and IVS4ins6kb, 334bp and 3541bp bands were detected in F1 / R1; 1437bp and 7494bp bands were detected in F2 / R2.

[0114] 2) When IVS16ins3kb was homozygous and IVS4ins6kb was heterozygous, a 3541bp band was detected in F1 / R1; and 1437bp and 7494bp bands were detected in F2 / R2.

[0115] 3) When IVS16ins3kb was heterozygous and IVS4ins6kb was homozygous, 334bp and 3541bp bands were detected in F1 / R1; and 7494bp band was detected in F2 / R2.

[0116] 4) When IVS4ins6kb homozygous and IVS4ins6kb homozygous, a 3541bp band was detected in F1 / R1; a 7494bp band was detected in F2 / R2.

[0117] In a sixth aspect of the present application, a method for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency-causing gene SLC25A13 is provided.

[0118] For the limitations of this method, please refer to the instructions for use of the kit described above.

[0119] Some examples are provided below.

[0120] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0121] Example 1 Primer design and its application

[0122] A primer set of 4 new primers was designed to detect the pathogenic genes of SLC25A13 IVS16ins3kb and IVS4ins6kb. For a schematic diagram of primer design, see Figure 1 and Figure 2 , primer information is shown in Table 1 below.

[0123] Table 1 Four SLC25A13 mutation detection primers and amplified fragment lengths

[0124]

[0125] application:

[0126] 1) Extraction of sample genomic DNA

[0127] Peripheral blood samples from patients with citrin deficiency and known IVS16ins3kb and IVS4ins6kb variants (8 IVS16ins3kb heterozygous samples, 5 IVS4ins6kb heterozygous samples, and 3 samples heterozygous for both IVS16ins3kb and IVS4ins6kb) were collected. Normal negative samples without citrin deficiency were used as controls. Genomic DNA was extracted using a conventional DNA extraction method (brand: Changchun Zhiang Biotechnology; product number: GO-BTCDs) to obtain genomic DNA at a concentration of 50 ng / μL-150 ng / μL.

[0128] 2) Multiplex LA-PCR amplification

[0129] LA-PCR amplification was performed using the genomic DNA extracted in step 1) above as a template. The primers used are listed in Table 1. Vazyme's Phanta enzyme (Brand: Vazyme; Catalog No.: P505) was used. The reaction system and reaction conditions are shown in Tables 2 and 3, respectively.

[0130] Table 2 50μL LA-PCR reaction system:

[0131]

[0132] Table 3 LA-PCR reaction conditions:

[0133]

[0134] 3) Agarose gel electrophoresis

[0135] The LA-PCR amplified products were electrophoresed in 1% ordinary agarose gel at 140V for 30 minutes, and the DNA bands were observed using a gel imaging system.

[0136] 4) Results

[0137] Eight SLC25A13 gene IVS16ins3kb and IVS4ins6kb negative samples all amplified 1437bp and 334bp bands, and the results were consistent with the known results. Figure 3 .

[0138] The 8 samples with heterozygous SLC25A13 gene IVS16ins3kb mutation all amplified 3541bp, 1437bp, and 334bp bands, and the results were consistent with the known results. Figure 4 .

[0139] The 5 samples with heterozygous SLC25A13 gene IVS4ins6kb mutation all amplified 7494bp, 1437bp, and 334bp bands, and the results were consistent with the known results. Figure 5 .

[0140] The three SLC25A13 gene IVS16ins3kb and IVS4ins6kb heterozygous variant samples all amplified 7494bp, 3541bp, 1437bp, and 334bp bands, which were consistent with the known results. Figure 6 .

[0141] The above implementation cases illustrate that the method of the present application can simultaneously detect at least two types of insertion variants in the SLC25A13 gene, namely IVS16ins3kb and IVS4ins6kb, through a single multiplex LA-PCR reaction. Although there are various techniques for detecting such variants, the most common techniques include LA-PCR, multiplex fluorescence quantitative PCR, and NGS sequencing, but they all have certain detection limitations. For example, LA-PCR uses long-fragment amplification primers designed and electrophoresis analysis of product size to determine variants. Generally, it can only amplify one variant and is complex to operate. Multiplex fluorescence quantitative PCR combines fluorescence quantitative PCR and multiplex PCR technologies to simultaneously detect multiple target sequences, but requires specific instruments and multiple fluorescent probes, which is costly and requires strict primer and probe design and reaction conditions. NGS sequencing detects genetic variants by analyzing split-read fragments and can identify mutation locations and single-base mutations, but requires professional bioinformatics analysis skills and software tools, is costly, and has uncertainty in detecting rare or novel mutations.

[0142] In contrast, the multiplex LA-PCR technique using primers designed according to one embodiment of this application offers significant advantages. It can simultaneously detect two insertion variants, IVS16ins3kb and IVS4ins6kb, in the SLC25A13 gene in a single reaction. This technique is simple to use, time-efficient, and low-cost, with minimal experimental reaction requirements. It offers improved timeliness and cost-effectiveness, meeting clinical needs.

[0143] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. A primer set for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency causative gene SLC25A13, characterized in that: The primer set comprises: Primer set 1: upstream and downstream primers with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 for amplifying IVS16ins3kb; and / or, Primer set 2: upstream and downstream primers with sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 for amplifying IVS4ins6kb.

2. A chip for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency-causing gene SLC25A13, characterized in that: It comprises the primer set according to claim 1.

3. Use of the primer set according to claim 1 or the chip according to claim 2 in preparing a kit for detecting mutations IVS16ins3kb and / or IVS4ins6kb of the citrin deficiency causative gene SLC25A13.

4. A kit, characterized in that It comprises the primer set according to claim 1 and / or the chip according to claim 2.

5. The kit according to claim 4, wherein The kit further comprises one or more of a nucleic acid releasing reagent, a nucleic acid extracting reagent, a DNA polymerase, dNTPs and a PCR buffer.

6. The kit according to claim 5, wherein The DNA polymerase includes one or more of Phanta enzyme and LA Taq enzyme.

7. The kit according to any one of claims 4 to 6, wherein The kit further comprises a positive quality control product and / or a negative quality control product; Optionally, the negative control does not contain the gene SLC25A13 with mutations IVS16ins3kb and IVS4ins6kb; Optionally, the positive control product contains the gene SLC25A13 with mutations IVS16ins3kb and IVS4ins6kb.

8. The method for using the kit according to any one of claims 4 to 7, wherein: It includes the following steps: extracting nucleic acid from the sample to be tested and preparing a nucleic acid sample; performing LA-PCR amplification on the nucleic acid sample; and, Obtain and analyze results; Optionally, the sample to be tested includes one or more of peripheral blood, tissue, saliva, oral swab and dried blood spot.

9. The method of use according to claim 8, wherein: In each reaction system for LA-PCR amplification: the initial system concentration of each primer is independently 5 μM to 15 μM, the initial system concentration of the nucleic acid sample is 50 ng / μL to 150 ng / μL, and the initial system concentration of dNTP is 5 mM to 15 mM; Optionally, the DNA polymerase in the kit is Phanta enzyme, and the amount of Phanta used is (0.5 μL~2 μL) / 50 μL reaction system.

10. The method of use according to claim 8 or 9, characterized in that: The procedure for performing LA-PCR amplification includes: Pre-denaturation, 95°C, 3 minutes; Denaturation, 95°C, 15 seconds, annealing, 60°C, 15 seconds, extension, 72°C, 4 minutes, 35 cycles; Extensive extension, 72°C, 4 minutes; Optionally, the LA-PCR amplification is long fragment amplification PCR.

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