A method for detecting the copy number of ndufaf2 gene based on multiple ligation probe amplification technology

Abstract

The application provides a method for detecting NDUFAF2 gene copy number based on multiple ligation probe amplification technology, and belongs to the technical field of biological detection. The application adopts multiple ligation probe amplification (MLPA) technology, and specific primer probes are designed for each exon and intron of the NDUFAF2 gene, and specific primer probes are also designed for the exons of the adjacent genes of the NDUFAF2 gene. The MLPA primer group can reliably detect the complex gene structure of the NDUFAF2 gene with large intron fragments and highly homologous sequences, has high detection efficiency, and is simple, fast and easy to operate. The application also prepares the MLPA primer group into a detection kit for NDUFAF2 gene copy number, detects the NDUFAF2 gene copy number, has high accuracy and specificity, reduces the risk of misdiagnosis and missed diagnosis, and has high repeatability, thereby providing more reliable guarantee for experimental results.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for detecting the copy number of the NDUFAF2 gene based on multiplex ligation probe amplification technology. Background Technology

[0002] The NDUFAF2 gene encodes NDUFAF2, a key protein in mitochondrial complex I that plays a crucial role in cellular energy metabolism. Mitochondrial complex I, also known as NADH:ubiquinone oxidoreductase, is a key complex in the mitochondrial respiratory chain, responsible for electron transport and proton pumping. The NDUFAF2 protein assists in the transfer of electrons from NADH to ubiquinone (coenzyme Q) in this process, playing an indispensable role in maintaining cellular energy homeostasis and preventing cellular aging.

[0003] However, dysfunction of the NDUFAF2 gene can lead to mitochondrial diseases, a group of inherited metabolic disorders that affect mitochondrial function and cellular energy production. In particular, mutations or abnormalities in the NDUFAF2 gene often result in abnormal assembly of mitochondrial complex I, affecting cellular energy metabolism and causing a variety of clinical manifestations, including neurological problems, heart disease, and muscle dysfunction. Mitochondrial diseases are typically inherited in an autosomal recessive manner, and it is estimated that approximately 1 in 8,500 people are affected. Mitochondrial complex I deficiency is the most common form of mitochondrial disease in children, accounting for about 30% of all cases.

[0004] The NDUFAF2 gene has unique structural features, including its four exons and a relatively large second intron (approximately 15 kb), as well as highly homologous sequences between exons 2 and 3. This makes deletions of this gene particularly common in pathogenic variants. Existing detection methods, such as whole-exome sequencing (WES) analysis, can identify pathogenic variants, but their accuracy and reliability are limited when faced with highly similar sequences, requiring further validation.

[0005] Traditional validation techniques, such as PCR-based methods and quantitative real-time PCR (Q-PCR), face numerous challenges. For example, the presence of large introns complicates breakpoint primer design, and Q-PCR methods typically require condition optimization and often employ dye methods with low reproducibility, limiting their applicability as first-line prenatal diagnostic methods. Summary of the Invention

[0006] This invention provides a method for detecting the copy number of the NDUFAF2 gene based on multiplexed probe amplification technology. The multiplexed probe set can accurately detect mitochondrial complex I deficiency caused by large fragment deletions of the NDUFAF2 gene, thereby improving the accuracy and reliability of the detection.

[0007] This invention provides a multiplexed probe set for detecting the copy number of the NDUFAF2 gene, including a probe set designed for all exons and introns of the NDUFAF2 gene, and a probe set designed for at least one exon of a neighboring gene of the NDUFAF2 gene.

[0008] Preferably, the nucleotide sequences of the probe set designed for exons 1, 2, 3 and 4 of the NDUFAF2 gene are shown in SEQ ID No. 1 to 8, respectively;

[0009] The nucleotide sequences of the probe set designed for introns 1 and 2 of the NDUFAF2 gene are shown in SEQ ID No. 9-12.

[0010] Preferably, the neighboring genes include the ERCC8 gene and the SMIM gene;

[0011] The nucleotide sequences of the probe set designed targeting exon 1 of the ERCC8 gene are shown in SEQ ID No. 13-14;

[0012] The nucleotide sequences of the probe set designed for exon 1 of the SMIM gene are shown in SEQ ID No. 15-16.

[0013] Preferably, it also includes universal primer probes, the nucleotide sequences of which are shown in SEQ ID No. 17-18.

[0014] The present invention also provides a multiplexed probe MIX, comprising a TE mixture of multiplexed probe groups shown in SEQ ID No. 1 to 16.

[0015] Preferably, in the multiple-connection probe MIX, the probe concentrations shown in SEQ ID No. 1-4 and SEQ ID No. 9-12 are all 16 nM, the probe concentrations shown in SEQ ID No. 5-8 are all 18 nM, and the probe concentrations shown in SEQ ID No. 13-16 are all 14 nM.

[0016] The present invention also provides the application of the above-mentioned multiplex probe set or the above-mentioned multiplex probe MIX in the preparation of a kit for detecting the copy number of the NDUFAF2 gene.

[0017] The present invention also provides a kit for detecting the copy number of the NDUFAF2 gene, comprising the above-mentioned multiplex ligation probe MIX, and further comprising a hybridization buffer, ligation buffer A, ligation buffer B, ligase, reference probe and universal probe.

[0018] This invention also provides a method for detecting the copy number of the NDUFAF2 gene for non-disease diagnostic purposes, comprising the following steps:

[0019] (1) Add 5 μL of DNA sample to each sample, treat the genomic DNA at 98℃ for 5 min, and use it as a template after denaturation at 25℃;

[0020] (2) Hybridization is performed by mixing the template described in step (1) with the hybridization buffer, reference probe and multiplex ligation probe MIX in the above kit; the hybridization procedure includes: holding at 95℃ for 1 min; holding at 60℃ for 16-18 h; and holding at 20℃.

[0021] (3) The hybridization product obtained in step (2) is mixed with ligation buffer A, ligation buffer B and ligase and then ligated. The ligation procedure includes: holding at 54℃ for 15 min; holding at 98℃ for 5 min; and holding at 20℃.

[0022] (4) Use the universal probe and the ligation product obtained in step (3) to prepare a PCR amplification system for PCR amplification. The PCR amplification program includes: 95℃ for 30s; 60℃ for 30s, 72℃ for 60s, 29 cycles; extension at 72℃ for 20min, and holding at 20℃.

[0023] (5) Use a capillary electrophoresis apparatus to detect the amplification products obtained in step (4) and then perform data analysis.

[0024] Preferably, step (5) includes analyzing the detection results using Coffalyser analysis software.

[0025] Beneficial effects: This invention employs multiplex ligation probe amplification (MLPA) technology, designing specific primers and probes for each exon and intron of the NDUFAF2 gene, and similarly designing specific primers and probes for exons of neighboring genes of the NDUFAF2 gene. The MLPA primer set described in this invention can reliably detect the complex gene structure of the NDUFAF2 gene, which has large introns and highly homologous sequences, with high detection efficiency and simple and rapid operation.

[0026] The present invention also prepares the MLPA primer set into a kit for detecting the copy number of the NDUFAF2 gene. When detecting the copy number of the NDUFAF2 gene, it has higher accuracy and specificity than traditional PCR and Q-PCR methods, reducing the risk of misdiagnosis and missed diagnosis; it has high repeatability, reducing inter-experimental variability and providing more reliable assurance for experimental results. Attached Figure Description

[0027] Figure 1This is the original capillary electrophoresis pattern of sample A obtained according to the method in Example 1; wherein, the dark bands marking regions 109-144 are the fragment regions detected by this invention, wherein in the detection of the target gene, band 116 is the detection band for exon 1 of the NDUFAF2 gene, band 123 is the detection band for exon 2 of the NDUFAF2 gene, band 144 is the detection band for exon 3 of the NDUFAF2 gene, and band 140 is the detection band for exon 4 of the NDUFAF2 gene; in the probe regions serving as controls, band 109 is the detection band for exon 1 of ERCC8, band 112 is the detection band for exon 1 of the SMIM gene, band 119 is the detection band for intron 1 of the NDUFAF2 gene, and band 126 is the detection band for intron 2 of the NDUFAF2 gene;

[0028] Figure 2 The sample copy number information is obtained by calculating the peak area after the test data of the same batch of sample A has been homogenized, and the test result is normal.

[0029] Figure 3 This is the peak area calculation information of the capillary electrophoresis pattern of sample A after homogenization;

[0030] Figure 4 This is the original capillary electrophoresis pattern of sample B obtained according to the method in Example 1;

[0031] Figure 5 The sample copy number information was obtained by calculating the peak area after the test data of the same batch of sample B was homogenized, and the test result was normal.

[0032] Figure 6 This is the peak area calculation information of the capillary electrophoresis pattern of sample B after normalization;

[0033] Figure 7 This is the original capillary electrophoresis pattern of sample C obtained according to the method in Example 1;

[0034] Figure 8 The sample copy number detection information is obtained by calculating the peak area after the test data of the same batch of sample C has been homogenized, and the test result is normal.

[0035] Figure 9 This is the peak area calculation information of the capillary electrophoresis pattern of sample C after normalization;

[0036] Figure 10 This is the original capillary electrophoresis pattern of sample D obtained according to the method in Example 2;

[0037] Figure 11The sample copy number detection information is obtained by calculating the peak area after the test data of the same batch of sample D has been homogenized, and the test result is normal.

[0038] Figure 12 This is the peak area calculation information of the capillary electrophoresis pattern of sample D after homogenization;

[0039] Figure 13 This is the original capillary electrophoresis pattern of sample E obtained according to the method in Example 2;

[0040] Figure 14 The sample copy number detection information is obtained by calculating the peak area after the test data of the same batch of sample E is homogenized. The test results show heterozygous deletion of exons 2, 3, and 4 of the NDUFAF2 gene and exon 1 of the SMIM15-1 gene.

[0041] Figure 15 This is the peak area calculation information of the capillary electrophoresis pattern of sample E after normalization;

[0042] Figure 16 This is the original capillary electrophoresis pattern of sample F obtained according to the method in Example 2;

[0043] Figure 17 The sample copy number detection information is obtained by calculating the peak area after the test data of the same batch of sample F is homogenized. The test results show that the first exon of NDUFAF2 gene and the first exon of ERCC8 gene are heterozygous deletions.

[0044] Figure 18 This is the peak area calculation information of the capillary electrophoresis pattern of sample F after homogenization. Detailed Implementation

[0045] This invention provides a multiplexed probe set for detecting the copy number of the NDUFAF2 gene, including a probe set designed for all exons and introns of the NDUFAF2 gene, and a probe set designed for at least one exon of a neighboring gene of the NDUFAF2 gene.

[0046] The NDUFAF2 gene described in this invention contains four exons and two introns. Probes are designed for all exons and introns in this structure to form an MLPA hybridization probe set for hybridization with a DNA library. The sequences of these probes are shown in SEQ ID Nos. 1-12. This invention also designs specific probes for exons of neighboring genes of the NDUFAF2 gene, preferably including the SMIM15 and ERCC8 genes, whose sequences are shown in SEQ ID Nos. 13-16. Preferably, this invention also designs universal primer probes for amplification of the probe set. The nucleotide sequence of the upstream probe of the universal primer is preferably shown in SEQ ID No. 17. Purification method: HPLC; 5' modification: none; sequence: GGGTTCCCTAAGGGTTGGA;

[0047] The preferred nucleotide sequence of the downstream probe of the universal primer is shown in SEQ ID No. 18. Purification method: HPLC; 5' modification: none; sequence: GTGCCAGCAAGATCCAATCTAGA.

[0048] The present invention also provides a multiplexed probe MIX, comprising a TE mixture of multiplexed probe groups shown in SEQ ID No. 1 to 16.

[0049] In the multiple connection probe MIX of the present invention, the information of each probe is preferably shown in Table 1.

[0050] Table 1. Sequences and concentrations of each probe in the MIX of this invention.

[0051]

[0052]

[0053] In this invention, all probes shown in SEQ ID Nos. 1 to 16 are preferably diluted to 10 μM using TE and then mixed to obtain the multi-linked probe MIX. The mixing described in this invention can be performed after grouping or directly.

[0054] In this embodiment of the invention, when preparing the multiplexed probe MIX, it is preferable to divide the 16 probes shown in SEQ ID No. 1 to 16 into three groups before mixing them, as follows:

[0055] The first group of probes consists of eight probes, namely SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12, mixed in a 1:1 ratio.

[0056] The second set of probes consists of four probes, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, mixed in a 1:1 ratio.

[0057] The third group of probes consists of four probes, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, mixed in a 1:1 ratio.

[0058] The three groups of probes were mixed and diluted in a volume ratio of Group 1: Group 2: Group 3: TE of 16:7:9:468 to prepare the multi-connection probe MIX.

[0059] The present invention also provides the application of the above-mentioned multiplex probe set or the above-mentioned multiplex probe MIX in the preparation of a kit for detecting the copy number of the NDUFAF2 gene.

[0060] The present invention can prepare the above-mentioned multiplexed probe set or multiplexed probe MIX into a kit for detecting the copy number of NDUFAF2 gene.

[0061] The present invention also provides a kit for detecting the copy number of the NDUFAF2 gene, comprising the above-mentioned multiplex ligation probe MIX, and further comprising a hybridization buffer, ligation buffer A (Ligase Buffer A), ligation buffer B (Ligase Buffer A), ligase, a reference probe, and a universal probe.

[0062] This invention does not specifically limit the source of other components in the kit besides the probe. For example, the hybridization buffer, ligation buffer A, ligation buffer B, ligase (Ligase-65 enzyme), and universal primers for PCR amplification are all from MRC-Holland. The reference probe in this invention is preferably SALS AMLPA Probemix P200 Reference-1 (referred to as p200 probe) purchased from Thermo Fisher Scientific, which is used as an internal standard to correct the bias caused by amplifying the product signal during the MLPA experiment and for subsequent data analysis.

[0063] The kit described in this invention may preferably also include a capillary electrophoresis apparatus and reagents, such as ionic formamide, Size Standard, and GeneScan, all available from Thermo Fisher Scientific. TM 500LIZ TM dye.

[0064] This invention also provides a method for detecting the copy number of the NDUFAF2 gene for non-disease diagnostic purposes, comprising the following steps:

[0065] (1) Add 5 μL of DNA sample to each sample, treat at 98℃ for 5 min, and use the denatured DNA sample at 25℃ as a template.

[0066] (2) Hybridization is performed by mixing the template described in step (1) with the hybridization buffer, reference probe and multiplex ligation probe MIX in the above kit; the hybridization procedure includes: holding at 95℃ for 1 min; holding at 60℃ for 16-18 h; and holding at 20℃.

[0067] (3) The hybridization product obtained in step (2) is mixed with ligation buffer A, ligation buffer B and ligase and then ligated. The ligation procedure includes: holding at 54℃ for 15 min; holding at 98℃ for 5 min; and holding at 20℃.

[0068] (4) Use the universal probe and the ligation product obtained in step (3) to prepare a PCR amplification system for PCR amplification. The PCR amplification program includes: 95℃ for 30s; 60℃ for 30s, 72℃ for 60s, 29 cycles; extension at 72℃ for 20min, and holding at 20℃.

[0069] (5) Use a capillary electrophoresis apparatus to detect the amplification products obtained in step (4) and then perform data analysis.

[0070] The present invention preferably uses a sample of genomic DNA, which is denatured by treating it at 98°C for 5 minutes, followed by indefinite incubation at 25°C. The genomic DNA used in this invention is preferably extracted from a blood sample.

[0071] This invention uses denatured DNA as a template to prepare a hybridization system comprising: 5 μL template (9–11 ng / μL), 1.5 μL hybridization buffer, 0.5 μL multiplex ligation probe MIX, and 1 μL p200 probe. After thoroughly mixing the hybridization system, a hybridization reaction is performed. The preferred procedure for the hybridization reaction includes: maintaining at 95°C for 1 min, then maintaining at 60°C for 16–18 h, and finally incubating at 20°C indefinitely.

[0072] This invention prepares a ligation system by combining the hybridization product with ligation buffer A, ligation buffer B, and ligase. The ligation system, in 50 μL units, preferably comprises: 8 μL of hybridization product, 25 μL of ultrapure water, 3 μL of Ligase Buffer A, 3 μL of Ligase Buffer B, and 1 μL of Ligase-65 enzyme. After thoroughly mixing the ligation system, ligation is performed. The preferred ligation procedure includes: incubation at 54°C for 15 min, followed by inactivation at 98°C for 5 min, and then incubation at 20°C indefinitely.

[0073] This invention utilizes universal primers to amplify the ligation product via PCR. The universal primers are a PCR Primer Mix containing two primer pairs at equal concentrations of 10 μM. The PCR amplification system consists of 40 μL of the ligation product, 7.5 μL of H2O, 2 μL of the PCR Primer Mix, and 0.5 μL of SALSA Polymerase. After thoroughly mixing the PCR amplification system, PCR amplification is performed. The preferred PCR amplification program includes: 29 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds, followed by a final extension at 72°C for 20 minutes, and then incubation at 20°C indefinitely to complete the amplification.

[0074] This invention preferably utilizes capillary electrophoresis to detect the amplification products. GeneScan preparation... TM 500LIZ TM Working solution: based on formamide dosage and GeneScan TM 500LIZ TM The dosage ratio is 10:0.2. After preparing the working solution, follow the GeneScan instructions. TM 500LIZ TM 10 μL of the working solution was mixed with 1.1 μL of the PCR product and electrophoresis was performed. The results were then analyzed using Coffalyser software. The calculated ratio (e.g.) was used to determine the final result. Figure 3 To confirm the presence of copy number variations in this gene, box plots (such as...) are used. Figure 2 The results are displayed.

[0075] To further illustrate the present invention, the following detailed description of a method for detecting the copy number of the NDUFAF2 gene based on multiplex ligation probe amplification technology provided by the present invention is provided in conjunction with embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0076] The experimental methods used in this embodiment of the invention are as follows:

[0077] 1. Custom probe MIX configuration

[0078] The 16 probes shown in SEQ ID No. 1 to 16, synthesized by the commissioned biotechnology company, were diluted to 10 μM using TE dilution.

[0079] (1) The first group of probes consists of eight probes, namely SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, and SEQ ID No.12, mixed in a 1:1 ratio.

[0080] (2) The second group of probes consists of four probes, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, mixed in a 1:1 ratio.

[0081] (3) The third group of probes consists of four probes, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, mixed in a 1:1 ratio.

[0082] (4) Mix the three probes in a volume ratio of 16:7:9:468 (Group 1:Group 2:Group 3:TE) and dilute them to prepare a self-designed probe MIX.

[0083] 2. Experimental Operation Procedure:

[0084] (1) Using genomic DNA samples, the PCR instrument was set to 98°C for 5 minutes, followed by denaturation at 25°C indefinitely.

[0085] (2) After vortexing, add 5 μL of each DNA denaturation product (diluted to 9-11 ng / μL) to 1 μL of ligation buffer and 0.5 μL of hybridization probe ProbeMix (custom probe MIX and 1 μL of p200 probe), mix well, set the hybridization program to 95℃ for 1 min, then 60℃ for 16-18 h, and finally incubate at 20℃ indefinitely.

[0086] (3) Use ligase buffer to prepare ligation system with Buffer A, ligation Buffer B and ligase Ligase-65enzyme. The ligation system is as follows: 25 μL water, 3 μL ligation Buffer A, 3 μL ligation Buffer B, 1 μL Ligase-65enzyme and 18 μL hybridization product. Set the PCR instrument ligation program to 54℃ for 15 min, then inactivate at 98℃ for 5 min, and finally incubate at 20℃ indefinitely.

[0087] (4) Prepare the PCR amplification system. The reaction system is as follows: 40 μL of ligation product, 7.5 μL of H2O, 2 μL of PCR Primer Mix, and 0.5 μL of SALSA Polymerase. The amplification program is 95℃ for 30 seconds, 60℃ for 30 seconds, and 72℃ for 60 seconds for a total of 29 cycles. Finally, extend the amplification at 72℃ for 20 minutes and then keep it at 20℃ indefinitely to complete the amplification.

[0088] (5) Detect the amplification products using a capillary electrophoresis apparatus. Prepare GeneScan. TM 500LIZ TM Working solution: based on formamide dosage and GeneScan TM 500LIZ TM The dosage ratio is 10:0.2. After preparing the working solution, follow the GeneScan instructions. TM 500LIZ TM Mix 10 μL of the working solution with 1.1 μL of the PCR product in (4) and perform an electrophoresis experiment.

[0089] (6) Analyze the results using the official Coffalyser analysis software.

[0090] Example 1

[0091] Stability detection

[0092] Three blood samples, A, B, and C, from healthy individuals from different families were collected. The experiment was conducted according to the above procedure, and the results of capillary electrophoresis were analyzed.

[0093] (1) The figure shows the original pattern detected by capillary electrophoresis. The capillary electrophoresis shows that all bands are detected normally. Regions 109-144 are the fragment regions detected by this invention. Among them, in the detection of the target gene, band 116 is the detection band of exon 1 of the NDUFAF2 gene, band 123 is the detection band of exon 2 of the NDUFAF2 gene, band 144 is the detection band of exon 3 of the NDUFAF2 gene, and band 140 is the detection band of exon 4 of the NDUFAF2 gene. In the probe region used as a control, band 109 is the detection band of exon 1 of ERCC8, band 112 is the detection band of exon 1 of the SMIM gene, band 119 is the detection band of intron 1 of the NDUFAF2 gene, and band 126 is the detection band of intron 2 of the NDUFAF2 gene.

[0094] (2) The original detection results of samples A, B, and C are respectively Figure 1 , Figure 4 , Figure 7The results show that all bands were detected and the test results are normal.

[0095] (3) The results were analyzed using software. The peak area of ​​all bands was calculated, and all reference genes were normalized. The normalized numerical results are shown in Appendix 3. Figure 6 , Figure 9 In the Ratio column, all test results were within the range of 1±0.1, indicating that the data were normal.

[0096] (4) The data results are presented using histograms, as follows: Figure 2 , Figure 5 , Figure 8 The results were all normal.

[0097] Example 2

[0098] Accuracy verification

[0099] Collect three samples from a family with a positive clinical sample: son (E), mother (F), and father (G). Perform the experiment according to the above procedure and analyze the results of capillary electrophoresis.

[0100] (1) Figure 10 , 13 The original spectrum from capillary electrophoresis in Figure 16 shows that all bands were detected, indicating that the detection experiment proceeded normally.

[0101] (2) The results show that, Figure 12 The neutron (E) detection results were normal. Figure 15 The results of the test on the mother (F) showed that exons 2, 3, and 4 of the NDUFAF2 gene and exon 1 of the SMIM15-1 gene were detected as heterozygous deletions, that is, the probe ratio of the gene was 0.5 ± 0.1. Figure 18 The results of the paternal (G) test showed that exon 1 of the NDUFAF2 gene and heterozygous deletion of exon 1 of the ERCC8 gene were detected, that is, the probe ratio value of the gene was 0.5±0.1.

[0102] (3) Figure 11 , Figure 14 , Figure 17 The displayed result is consistent with the ratio value.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Detection NDUFAF2 A multiplexed probe set for gene copy number determination, characterized in that, The multi-connection probe group is for: targeting NDUFAF2 Probe sets designed for all exons and introns of the gene and targeting the NDUFAF2 Probe sets designed from the exons of neighboring genes; against NDUFAF2 The nucleotide sequences of the probe sets designed for exons 1, 2, 3 and 4 of the gene are shown in SEQ ID No. 1~8, respectively; against NDUFAF2 The nucleotide sequences of the probe sets designed for introns 1 and 2 of the gene are shown in SEQ ID No. 9~12, respectively; The neighboring genes are ERCC8 Genes and SMIM15 Gene; against ERCC8 The nucleotide sequences of the probe set designed for exon 1 of the gene are shown in SEQ ID No. 13~14; against SMIM15 The nucleotide sequences of the probe set designed for exon 1 of the gene are shown in SEQ ID No. 15~16.

2. The multiple connection probe assembly according to claim 1, characterized in that, It also includes universal primer probes, the nucleotide sequences of which are shown in SEQ ID No. 17~18.

3. A multi-connection probe MIX, characterized in that, TE mixture including the multiple-linked probe groups shown in SEQ ID No. 1~16.

4. The multiple connection probe MIX according to claim 3, characterized in that, In the multiple-connection probe MIX, the probe concentrations shown in SEQ ID No. 1~4 and SEQ ID No. 9~12 are all 16 nM, the probe concentrations shown in SEQ ID No. 5~8 are all 18 nM, and the probe concentrations shown in SEQ ID No. 13~16 are all 14 nM.

5. The multiplexed probe assembly of claim 1 or 2, or the multiplexed probe MIX of claim 3 or 4, in the preparation and detection of... NDUFAF2 Applications in gene copy number assay kits.

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