WO2023179053A1 - 一种用于靶基因拷贝数检测的方法 - Google Patents

一种用于靶基因拷贝数检测的方法 Download PDF

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WO2023179053A1
WO2023179053A1 PCT/CN2022/131962 CN2022131962W WO2023179053A1 WO 2023179053 A1 WO2023179053 A1 WO 2023179053A1 CN 2022131962 W CN2022131962 W CN 2022131962W WO 2023179053 A1 WO2023179053 A1 WO 2023179053A1
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smn1
gene
internal reference
gapdh
reference gene
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French (fr)
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钱学庆
秦炜
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上海润达榕嘉生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions

  • the present application belongs to the field of gene detection technology, and specifically relates to a method for target gene copy number detection and its primer-probe combination.
  • SMA Spinal Muscular atrophy
  • Type IV onset age ranges from 15 to 60 years old. The onset and progression are both insidious, but the prognosis is relatively good, and walking ability can often be maintained throughout life 1 .
  • SMA Survival Motor Neuron 1
  • This gene is located in the chromosome 5q13.2 region, and its product SMN protein is an important protein in maintaining the normal physiological functions of bones and intercostal muscles 2 .
  • About 95% of SMA patients have deletion mutations in both copies of the SMN1 gene, resulting in a lack of normal functioning SMN protein in the body and causing the disease.
  • SMN1 SMN2
  • SMN2 is highly homologous to SMN1, with only 5 nucleotide differences between them. Four of them are located in non-coding regions, and only one (c.840C>T) is located in the coding region 2 of exon 7 of the SMN2 gene.
  • the base change in the coding region does not change the amino acid sequence of the protein, but it can affect the splicing of exons, causing 50-90% of the SMN proteins produced to be abnormally spliced and lose biological activity 4 .
  • the SMN2 gene can still produce a certain amount of SMN protein with biological functions, compensatingly alleviating the symptoms caused by SMN1 deletion to a certain extent through dose compensation. Therefore, clinically, the increase in SMN2 gene copy number in some SMA patients can alleviate the symptoms of the disease to a certain extent 5 .
  • PCR-RFLP polymerase chain restriction fragment length polymorphism
  • MPA Multiplex Ligation-dependent Probe Amplification
  • Real-time fluorescence quantitative PCR mainly analyzes the Ct value in PCR amplification of target genes and internal reference genes, and analyzes the target gene copy number through the ⁇ Ct method. This method is mostly used to analyze gene expression levels in biology. When the differences in expression levels between genes are large, especially when the differences are exponential, this method is more reliable. However, among SMA carriers, most of them have one copy of SMN1, which is only twice as different as the two copies of normal individuals, and the difference in Ct value is not very significant. It is precisely because the fluorescence quantitative PCR method cannot accurately distinguish heterozygous deletion carriers from wild-type normal individuals.
  • the present application provides a method for detecting the copy number of human motor neuron survival gene SMN1 and its primer-probe combination.
  • the Rn value (Normalized reporter) reflects the fluorescence intensity of each cycle, and the difference ⁇ Rn between it and the baseline background fluorescence value (Rn-) can represent the amount of PCR product in each amplification cycle.
  • Various components in the PCR reaction such as template concentration, primer concentration, dNTP concentration, Mg 2+ concentration, etc., will affect the product amplification efficiency.
  • the gene to be tested SMN1 and the internal reference gene GAPDH are simultaneously amplified in the PCR amplification system, and there is a competitive relationship between the two.
  • this application reduces the dNTP concentration in the reaction from the normal 200 ⁇ M to less than 80 ⁇ M to limit the amplification efficiency of the test gene SMN1 and the internal reference gene GAPDH.
  • the gene to be tested SMN1 and the internal reference gene GAPDH both have 2 copy numbers, and the concentrations of the two genes are the same (1:1), the competition between the two genes for the restricting reagent dNTP in the reaction will reach an equilibrium level, and the amplification products of the two genes will reach an equilibrium level. A level of balance will also be reached.
  • the template concentration ratio between the two is 1:2.
  • the template concentration of the gene SMN1 to be tested is relatively reduced, and its competition level in the amplification system is reduced. Therefore, the reaction reagent dNTP obtained through competition is also reduced accordingly, the PCR amplification product is reduced, and the ⁇ Rn value is reduced.
  • the competitive ability of the internal reference gene GAPDH is relatively enhanced, and the reaction reagent dNTP obtained by it will also increase accordingly, the PCR amplification product will increase, and the ⁇ Rn value will increase.
  • this application provides a method for detecting the copy number of a target gene, which method includes:
  • Target gene copy number analysis Calculate the ratio C of the ⁇ Rn value of the amplification curve of the target gene and the internal reference gene, and determine the target gene copy number based on the ratio C.
  • the target gene is SMN1 and the internal reference gene is GAPDH.
  • the criteria for judging the target gene copy number include:
  • the ratio C of the ⁇ Rn value of the amplification curve of SMN1 and the internal reference gene GAPDH is 0.85 to 1.15, then it is judged that both the SMN1 gene and the internal reference gene GAPDH have 2 copies;
  • the ratio C of the ⁇ Rn value of the amplification curve of SMN1 and the internal reference gene GAPDH is 0.3 to 0.7, then it is judged that the copy number of the SMN1 gene is 1 copy and the internal reference gene GAPDH is 2 copies; or
  • the ratio C of the ⁇ Rn value of the amplification curve of SMN1 and the internal reference gene GAPDH is -0.1 to 0.1, then the copy number of the SMN1 gene is judged to be zero copies.
  • step (2) specifically includes the following steps:
  • the primer-probe combination for detecting the target gene includes a primer-probe combination for detection of copy number of human motor neuron survival gene SMN1, and the primer-probe combination for detection of copy number of human motor neuron survival gene SMN1
  • the combination includes primer pairs and probes that specifically amplify and detect the human motor neuron survival gene SMN1;
  • the primer pair that specifically amplifies the human motor neuron survival gene SMN1 includes:
  • Reverse primer SEQ ID No:2 GCTGGCAGACTTACTCCTTA;
  • the probe for detecting the human motor neuron survival gene SMN1 includes the nucleotide sequence shown in SEQ ID No: 3: AGAAGGAAGGTGCTCACATT.
  • the 5' end of the probe is modified with a fluorescent dye
  • the 3' end of the probe is modified with a quencher.
  • the 3' end of the probe is also connected to an MGB group.
  • the fluorescent dye includes FAM.
  • the quencher includes TAMRA.
  • the designed MGB probe primer uses FAM fluorescent dye to modify the 5' end, which has strong fluorescence signal and the highest detection efficiency.
  • NCBI code of the SMN1 gene is GenBank: NG_008691.1.
  • the forward primer and the reverse primer correspond to bases 31985-32006 and 32049-32068 of the SMN1 gene respectively, and the MGB probe corresponds to SMN1 gene 32021. ⁇ 32040 bases.
  • the primer-probe combination for detecting the internal reference gene includes a primer pair and a probe for specifically amplifying and detecting the internal reference gene GAPDH;
  • the primer pair for specifically amplifying the internal reference gene GAPDH includes:
  • Reverse primer SEQ ID No:5 CTCCCTTGAGCTTCCCTGC;
  • the probe for detecting the internal reference gene GAPDH includes the nucleotide sequence shown in SEQ ID No: 6: TTGGGCAGCCCTGGA.
  • the 5' end of the probe is modified with a fluorescent dye
  • the 3' end of the probe is modified with a quencher.
  • the 3' end of the probe is also connected to an MGB group.
  • the fluorescent dye includes any one of FAM, TET, VIC or HEX.
  • the quencher includes BHQ.
  • the GAPDH gene is used as the internal reference gene, which has good amplification effect and high detection efficiency.
  • the forward primer and reverse primer designed in this application have random base distribution. There is no continuous G or C base aggregation at the 3' end of the primer, and there is no self-complementary overlapping sequence at the 3' end, which can avoid hairpin structures and primer duplication. The production of aggregates.
  • the forward primer is specially designed at the C>T difference base at the c.840 position in exon 7 of the SMN1 gene and SMN2 gene, which can prevent the SMN2 gene from interfering with the amplification of the SMN1 gene.
  • the concentration of the sample DNA is 5-100ng/ ⁇ L, for example, it can be 5ng/ ⁇ L, 10ng/ ⁇ L, 20ng/ ⁇ L, 30ng/ ⁇ L, 40ng/ ⁇ L, 50ng/ ⁇ L, 60ng/ ⁇ L, 70ng/ ⁇ L , 80ng/ ⁇ L, 90ng/ ⁇ L or 100ng/ ⁇ L, etc.
  • the final concentrations of the forward primer and the reverse primer that specifically amplify the human motor neuron survival gene SMN1 in the PCR system are independently 0.1 to 1 ⁇ M, for example, they can be 0.1 ⁇ M, 0.3 ⁇ M, or 0.5 ⁇ M, 0.7 ⁇ M, 0.9 ⁇ M or 1 ⁇ M, etc.
  • the final concentrations of the forward primer and the reverse primer that specifically amplify the internal reference gene GAPDH in the PCR system are independently 0.1 to 1 ⁇ M, for example, they can be 0.1 ⁇ M, 0.3 ⁇ M, 0.5 ⁇ M, or 0.7 ⁇ M. , 0.9 ⁇ M or 1 ⁇ M, etc.
  • the final concentrations of the probe for detecting the human motor neuron survival gene SMN1 and the probe for detecting the internal reference gene GAPDH in the PCR system are independently 0.1 to 1 ⁇ M, for example, they can be 0.1 ⁇ M, 0.3 ⁇ M, 0.5 ⁇ M, 0.7 ⁇ M, 0.9 ⁇ M or 1 ⁇ M, etc.
  • the final concentration of dNTP in the PCR system is 80-1.0 ⁇ M, for example, it can be 80 ⁇ M, 60 ⁇ M, 40 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5.0 ⁇ M or 1.0 ⁇ M, etc.
  • the final concentration of DNA polymerase in the PCR system is 0.02-0.1U/ ⁇ L, for example, it can be 0.02U/ ⁇ L, 0.03U/ ⁇ L, 0.05U/ ⁇ L, 0.07U/ ⁇ L, 0.09U/ ⁇ L or 0.1U/ ⁇ L, etc.
  • the PCR amplification procedure includes:
  • Pre-denaturation incubate at 93-96°C for 3-7 minutes.
  • the incubation temperature can be, for example, 93°C, 94°C, 95°C or 96°C.
  • the incubation time can be, for example, 3min, 4min, 5min, 6min or 7min, etc.;
  • Denaturation Incubate at 93-96°C for 25-35s.
  • the incubation temperature can be, for example, 93°C, 94°C, 95°C or 96°C.
  • the incubation time can be, for example, 25s, 27s, 29s, 30s, 31s, 33s or 35s, etc. ;
  • the incubation temperature can be, for example, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C.
  • the incubation time can be, for example, 85s, 87s, 89s, 90s, 91s, 93s or 95s, etc.;
  • the incubation temperature can be, for example, 70°C, 71°C, 72°C, 73°C or 74°C.
  • the incubation time can be, for example, 25s, 27s, 29s, 30s, 31s, 33s or 35s. wait;
  • Cycle 38 to 42 times The number of cycles may be, for example, 38, 39, 40, 41 or 42 times;
  • Warmth preservation Storage at 0 ⁇ 4°C, for example, it can be 0°C, 1°C, 2°C, 3°C or 4°C, etc.
  • the method further includes the step of using quality control reagents for simultaneous detection.
  • the target gene SMN1 and the internal reference gene GAPDH of the individual to be tested are simultaneously amplified in the same reaction, and a copy number calculation method is creatively designed to calculate the ⁇ Rn ratio of the amplification curves of the two on the qPCR amplification curve.
  • the judgment method of this application does not require the preparation of a qPCR quantitative standard curve, and is simple to operate and easy to use. The results are accurate and are conducive to widespread application.
  • the method of use further includes the step of determining the deletion of the SMN1 gene.
  • the criteria for judging the deletion of the SMN1 gene include:
  • Both the SMN1 gene and the internal reference gene GAPDH have 2 copies, and the SMN1 gene is wild type;
  • the copy number of the SMN1 gene is 1 copy, the internal reference gene GAPDH is 2 copies, and the SMN1 gene is a heterozygous deletion type; or
  • the copy number of the SMN1 gene is zero copies, there is no amplification product of SMN1, and the SMN1 gene is a homozygous deletion type.
  • the present application provides a kit for detecting the copy number of the human motor neuron survival gene SMN1.
  • the kit for detecting the copy number of the human motor neuron survival gene SMN1 includes the detection method described in the first aspect.
  • the reaction reagents include sample DNA extraction reagents and detection system PCR reaction solution.
  • the PCR reaction solution of the detection system includes DNA polymerase, enzyme buffer, MgCl 2 and dNTPs.
  • the quality control reagents include SMN1 zero-copy quality control product, SMN1 single-copy quality control product or SMN1 two-copy quality control product.
  • the SMN1 zero-copy quality control product is a plasmid containing two copies of the internal reference gene GAPDH.
  • the SMN1 single copy quality control product is a plasmid containing a single copy of the SMN1 gene and two copies of the internal reference gene GAPDH.
  • the two-copy SMN1 quality control product is a plasmid containing two copies of the SMN1 gene and two copies of the internal reference gene GAPDH.
  • the kit for detecting the copy number of human motor neuron survival gene SMN1 is designed with forward primers, reverse primers and MGB probe primers for detecting SMN1, which can be used for fluorescence quantitative PCR and other PCRs at the same time.
  • the amplification efficiency of the primers is high, and qPCR detection of plasmid standards and submitted samples can accurately detect and determine the patient's SMN1 genotype.
  • the reaction conditions such as forward primer, reverse primer, MGB probe concentration and annealing temperature, the amplification efficiency can be optimized.
  • the method of using the kit for detecting the copy number of human motor neuron survival gene SMN1 for the purpose of non-disease diagnosis and/or treatment includes the following steps:
  • the concentration of the DNA sample to be tested is 5 to 100 ng/ ⁇ L
  • the final concentrations of the forward primer and the reverse primer that specifically amplify the human motor neuron survival gene SMN1 are independently 0.1 to 1 ⁇ M
  • the final concentrations of the forward primer and the reverse primer for amplifying the internal reference gene GAPDH are each independently 0.1 to 1 ⁇ M
  • the final concentrations of the probe for detecting the human motor neuron survival gene SMN1 and the probe for detecting the internal reference gene GAPDH are independently independent.
  • the final concentration of dNTP in the PCR system is 80-1.0 ⁇ M
  • the final concentration of DNA polymerase in the PCR system is 0.02-0.1 U/ ⁇ L.
  • the PCR amplification procedure includes:
  • Pre-denaturation incubate at 93-96°C for 3-7 minutes;
  • Annealing and extension incubate at 50 ⁇ 55°C for 85 ⁇ 95s, incubate at 70 ⁇ 74°C for 25 ⁇ 35s, cycle 38 ⁇ 42 times;
  • Insulation Store at 0 ⁇ 4°C.
  • Both the SMN1 gene and the internal reference gene GAPDH have 2 copies.
  • the ratio C between the amplification curves ⁇ Rn of SMN1 and the internal reference gene GAPDH is at a normal level (0.85-1.15 range).
  • the SMN1 gene is wild type;
  • the copy number of the SMN1 gene is 1 copy, and the internal reference gene GAPDH is 2 copies.
  • the ratio C between the amplification curves ⁇ Rn of SMN1 and the internal reference gene GAPDH is significantly reduced (0.3-0.7 range), and the SMN1 gene is a heterozygous deletion. type; or
  • the copy number of the SMN1 gene is zero copies, and there is no amplification product of SMN1.
  • the ratio C between the amplification curve ⁇ Rn of SMN1 and the internal reference gene GAPDH is 0 (range of -0.1 to +0.1).
  • the SMN1 gene is a homozygous deletion type. .
  • the present application provides a system for detecting copy number of human motor neuron survival gene SMN1.
  • the system for detecting deletion of human motor neuron survival gene SMN1 includes:
  • Sample preparation module Collect and process the DNA sample to be tested, add the DNA sample to be tested, and the primer probe combination for SMN1 and internal reference gene GAPDH copy number detection into the detection system PCR reaction solution, mix, and prepare the PCR system;
  • Detection module perform amplification detection on the PCR system to obtain amplification curves of SMN1 and GAPDH;
  • Analysis module Calculate the ⁇ Rn value of the amplification curves of SMN1 and GAPDH, and perform SMN1 gene copy number analysis;
  • the primer-probe combination for detecting the copy number of SMN1 and the internal reference gene GAPDH, the PCR system, and the method for judging the copy number of the SMN1 gene are as described in the first and second aspects above.
  • the detection method designed in this application can effectively detect the copy number of the SMN1 gene.
  • the detection method does not require the preparation of a quantitative standard curve, is simple to operate, and is easy to use. According to the range of the ⁇ Rn ratio, the C value can effectively The copy number of the SMN1 gene was detected, and the three genotypes of homozygous deletion, heterozygous deletion and wild type were further analyzed;
  • the forward and reverse amplification primers for detecting the SMN1 gene in this application can efficiently amplify the target gene, have high specificity and accuracy, and have high detection efficiency.
  • the specificity of the MGB probe Good stability, strong fluorescence signal, and high detection efficiency;
  • the qPCR amplification curve analysis technology has the advantages of simple and convenient operation, easy to judge the results, low cost and high throughput. It will greatly improve the detection efficiency of SMN1 gene heterozygous deletion carriers and be effectively used to evaluate SMN1 gene detection reagents.
  • the performance of the cassette, research on the mechanisms related to the SMN1 gene, and the intermediate result information of auxiliary analysis to detect SMN1 gene deletion are of great significance to the field of spinal muscular atrophy detection.
  • the method developed in this application can not only be used to detect the copy number of SMN1 gene, but can also be used to detect the copy number of other genes.
  • Figure 1 is a graph showing the detection results of two copies of the SMN1 gene quality control product in Test Example 1.
  • Figure 2 is a graph showing the detection results of the SMN1 gene single copy quality control product in Test Example 1.
  • Figure 3 is a graph showing the detection results of the SMN1 gene zero-copy quality control product in Test Example 1.
  • Figure 4 is a graph showing the detection results of the two copy numbers of the SMN1 gene in the wild-type sample in Test Example 2.
  • Figure 5 is a graph showing the detection results of the single copy number of the SMN1 gene in the heterozygous deletion sample in Test Example 2.
  • Figure 6 is a graph showing the detection results of the two copy numbers of the SMN1 gene in the homozygous deletion sample in Test Example 2.
  • This embodiment provides a primer-probe combination for detecting the copy number of the human motor neuron survival gene SMN1.
  • the primer-probe combination for detecting the copy number of the human motor neuron survival gene SMN1 includes specific amplification and detection.
  • the nucleotide sequence of the forward primer that specifically amplifies the human motor neuron survival gene SMN1 is shown in SEQ ID No: 1, and the nucleotide sequence of the reverse primer is shown in SEQ ID No: 2; the detection method
  • the nucleotide sequence of the probe of the human motor neuron survival gene SMN1 is shown in SEQ ID No: 3.
  • the 5' end of the probe is modified with FAM, and the 3' end of the probe is modified with TAMRA.
  • the MGB group is also attached to the 3' end of the needle.
  • the nucleotide sequence of the forward primer for specifically amplifying the internal reference gene GAPDH is shown in SEQ ID No: 4, and the nucleotide sequence of the reverse primer is shown in SEQ ID No: 5; detecting the internal reference gene GAPDH
  • the nucleotide sequence of the probe is shown in SEQ ID No: 6.
  • the 5' end of the probe is modified with HEX, the 3' end of the probe is modified with BHQ, and the 3' end of the probe is also connected. MGB group.
  • SEQ ID No:1 TTTATTTTCCTTACAGGGTTTC.
  • SEQ ID No:2 GCTGGCAGACTTACTCCTTA.
  • SEQ ID No:3 AGAAGGAAGGTGCTCACATT.
  • SEQ ID NO:4 AAGGGCTTCGTATGACTGGG.
  • SEQ ID NO:5 CTCCCTTGAGCTTCCCTGC.
  • SEQ ID NO:6 TTGGGCAGCCCTGGA.
  • This embodiment provides a kit for detecting the copy number of human motor neuron survival gene SMN1.
  • the kit for detecting the copy number of human motor neuron survival gene SMN1 includes the kit for human motor neuron survival gene SMN1 copy number detection described in Example 1. Primer probe combinations, reaction reagents and quality control reagents for detection of motor neuron survival gene SMN1 copy number.
  • the reaction reagents include sample DNA extraction reagent (capgemini extraction kit, item number 51104), ethanol and detection system PCR reaction solution.
  • the detection system PCR reaction solution includes DNA polymerase, enzyme buffer, MgCl 2 and dNTPs .
  • the quality control reagents include SMN1 two-copy quality control, SMN1 single-copy quality control or SMN1 zero-copy quality control.
  • the SMN1 two-copy quality control is a plasmid containing two copies of the SMN1 gene and two copies of the internal reference gene GAPDH.
  • the SMN1 single-copy quality control is a plasmid containing a single copy of the SMN1 gene and two copies of the internal reference gene GAPDH.
  • the SMN1 zero-copy plasmid The control is a plasmid containing two copies of the internal reference gene GAPDH.
  • the above quality control plasmids were synthesized by Shanghai Bioengineering Co., Ltd.
  • This test example uses the kit for human motor neuron survival gene SMN1 copy number detection described in Example 2 to test the amplification curves of SMN1 zero-copy quality control, SMN1 single-copy quality control and SMN1 two-copy quality control.
  • the PCR system is as follows:
  • SMN1 zero-copy quality control SMN1 single-copy quality control and SMN1 two-copy quality control were added for detection.
  • PCR amplification was performed on the ABI7500 PCR instrument. The procedure of PCR amplification is as follows:
  • Pre-denaturation incubate at 94°C for 5 minutes;
  • Annealing and extension incubate at 55°C for 90 seconds, incubate at 72°C for 30 seconds, cycle 40 times;
  • Insulation Store at 4°C.
  • test results are shown in Figure 1, Figure 2 and Figure 3.
  • the test results of the quality control of two copies of the SMN1 gene are shown in Figure 1.
  • the test results of the quality control of the single copy of the SMN1 gene are shown in Figure 2.
  • the test results of the SMN1 gene zero
  • the test results of copy quality control are shown in Figure 3.
  • this application creatively designed a copy number calculation method based on qPCR, which can quickly and accurately analyze the SMN1 gene copy number without the need to prepare a qPCR quantitative standard curve. It is simple to operate and easy to use.
  • This test example uses the kit for human motor neuron survival gene SMN1 copy number detection described in Example 2 to detect clinical specimens and evaluate the accuracy of the kit.
  • the clinical specimens include 24 anticoagulant blood specimens. .
  • the PCR system is as follows:
  • the sample DNA template is genomic DNA extracted from whole blood samples, and the final concentration of the genomic DNA in the PCR system is 50 ng/ ⁇ L. Prepare PCR reaction solution according to the number of test copies.
  • PCR amplification was performed on the ABI7500 PCR instrument. The procedure of PCR amplification is as follows:
  • Pre-denaturation incubate at 94°C for 5 minutes;
  • Annealing and extension incubate at 55°C for 90 seconds, incubate at 72°C for 30 seconds, cycle 40 times;
  • Insulation Store at 4°C.
  • the criteria for judging the deletion of the SMN1 gene include:
  • Both the SMN1 gene and the internal reference gene GAPDH have 2 copies.
  • the ratio C between the amplification curves ⁇ Rn of SMN1 and the internal reference gene GAPDH is in the range of 0.85-1.15.
  • the SMN1 gene is wild type;
  • the copy number of the SMN1 gene is zero copies, and there is no amplification product of SMN1.
  • the ratio C between the amplification curve ⁇ Rn of SMN1 and the internal reference gene GAPDH is 0 (range of -0.1 to +0.1).
  • the SMN1 gene is a homozygous deletion type. .
  • the detection results are shown in Figure 4, Figure 5 and Figure 6.
  • the detection results of the wild-type sample with two copies of the SMN1 gene are shown in Figure 4, and the detection results of the heterozygous deletion sample with a single copy of the SMN1 gene are shown in Figure 5.
  • Figure 6 shows the detection results of samples with homozygous deletion of zero copy of the SMN1 gene.
  • the qPCR amplification curve ratio detection results of the above 24 samples showed that there were 4 wild-type (two copies) samples, 10 heterozygous deletion (single copy) samples, and 10 homozygous deletion (zero) samples. copy) sample, and is consistent with the copy number results of SMN1 clinical detection, with an accuracy of 100%.
  • the method for detecting the deletion of human motor neuron survival gene SMN1 and its primer-probe combination described in this application have good specificity and sensitivity.
  • the amplification curve ratio detection method of deletion detection has high detection accuracy, good amplification effect, simple operation, easy to judge the results, low detection cost and high throughput, which greatly improves the detection efficiency of SMN1 gene heterozygous deletion carriers. , which is of great significance to the field of spinal muscular atrophy detection.
  • the method developed in this application can not only be used to detect the copy number of SMN1 gene, but can also be used to detect the copy number of other genes.

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Abstract

本申请提供一种用于靶基因拷贝数检测的方法，在同一反应中同时扩增待测样本的靶基因和内参基因，通过计算qPCR扩增曲线上两者的扩增曲线ΔRn比值C(C＝ΔRn 靶基因/ΔRn 内参基因)确定待测个体的靶基因拷贝数，根据该比值的范围可有效检测出靶基因的基因型。

Description

一种用于靶基因拷贝数检测的方法 技术领域
本申请属于基因检测技术领域，具体涉及一种用于靶基因拷贝数检测的方法及其引物探针组合。
背景技术
脊髓性肌萎缩症(Spinal Muscular atrophy，SMA)是一种由于脊髓前角运动神经元退化引起的神经肌肉性疾病。本病为先天性隐形遗传病，多发于新生儿。患儿病情轻重程度不同，按照发病年龄和表型分为四型：I型，出生后6个月内发病，表现为肌张力低下，肌无力以四肢近端肌群受累为主，躯干肌亦无力；II型，通常于1岁内起病，婴儿早期生长正常，但6个月以后运动发育迟缓，虽然能坐，但独站及行走均未达到正常；III型，多数于5岁前起病，表现为进行性肢体近端肌无力和萎缩；IV型，发病年龄为15～60岁，起病和进展均较隐袭，但预后相对较好，行走能力常可保持终生 1。
所有类型的SMA均由运动神经元存活基因1(Survival Motor Neuron 1，SMN1)发生的纯合缺失导致。该基因位于染色体5q13.2区域，其产物SMN蛋白是维持骨骼和肋间肌正常生理功能的重要蛋白 2。约95％的SMA患者体内SMN1基因的2个拷贝均发生了缺失突变，导致体内缺乏正常功能的SMN蛋白，引起发病。也有少数病人是由于1个SMN1基因拷贝发生缺失，而另一个SMN1基因拷贝中出现了其它突变，也能导致体内丧失正常功能的SMN蛋白，引起发病 3。
在SMN1基因所在的区域内还存在着另一个SMN1的同源基因SMN2。SMN2与SMN1高度同源，两者间只存在5个核苷酸的差异。其中4个位于非编码区，只有1个(c.840C>T)位于SMN2基因第7号外显子的编码区 2。该编码区的碱基改变并不改变蛋白的氨基酸序列，但却能影响外显子的剪接，导致其生成的50-90％的SMN蛋白出现异常剪接，丧失生物学活性 4。尽管如此，SMN2基因还是能够生成一定数量的具有生物学功能的SMN蛋白，在一定程度上通过剂量补偿作用代偿性减轻SMN1缺失引起的症状。因此，临床上部分SMA患者SMN2基因拷贝数的增加可以一定程度上缓解疾病的症状 5。
脊髓性肌萎缩症作为一种常染色体隐性遗传病是世界范围内新生儿死亡的重要遗传因素之一 6。已报道文献中，世界各地不同人群中携带者频率约为1:47到1:72，活产儿中发病比例约为6000-10000分之一 7。由于本病可严重威胁患者的生命健康，同时人群中携带者比例较高，孕前携带者筛查可通过遗传咨询有效降低新生儿中的发病率 8。由于SMN1缺失是SMA发病的最主要原因，携带者筛查主要是检测SMN1拷贝数的变异 9。
目前已报道的SMA基因诊断方法众多，不同的方法的稳定性、可靠性、操作难易程度及经济成本等指标各不相同。这些诊断方法中聚合酶链式限制性片段长度多态性分析(PCR-restriction fragment length polymorphism，PCR-RFLP)可靠性最高，可作为基因诊断的“金标准”，但其操作步骤复杂，需要琼脂糖电泳，EB染色等步骤，无法实现大规模检测，也不符合体外诊断产品注册的要求。多重探针连接扩增技术(Multiplex Ligation-dependent Probe Amplification，MLPA)通过对不同靶区域的扩增，利用毛细管电泳检测各个区域产物信号强度，再将受检个体样本与正常对照样本比较，检测受检个体目标区域拷贝数的变异。其准确性、可靠性较高，但需毛细管电泳操作，步骤繁琐，对设备、人员要求都非常高，极大地限制了该方法的推广。
实时荧光定量PCR(real-time PCR)主要是通过对靶基因和内参基因PCR扩增中Ct值的分析，通过 ΔΔCt方法分析靶基因拷贝数。该方法多用于生物学中基因表达水平分析，当基因间表达水平差异较大，特别是呈指数差异时，该方法可靠性较高。但在SMA携带者中，其SMN1多为1个拷贝，与正常个体的2个拷贝仅相差一倍，在Ct值上差别并不十分显著。某些正是由于荧光定量PCR方法不能准确区分杂合缺失携带者与野生型正常个体，目前国家药品监督管理局只批准了该方法用于纯合突变的检测，尚不能用于杂合缺失突变检测。此外，芯片杂交、SNP分析、高通量测序等新技术也都可以应用于拷贝数分析，但其技术平台复杂，成本过高，不适合大规模临床推广。
Luming Zhou等在2015年报道的通过对产物溶解曲线的定量分析可有效检出杂合缺失携带者 11。之后，国内又有研究者在此基础上进一步改进、补充了检测步骤和分析方法 12溶解曲线分析方法一定程度上可以检测SMN1基因拷贝数，但最大的问题是PCR过程不直观，不能实时监测反应进程。并且该方法特异性不高，终产物中如有片段大小类似的非特异性扩增产物也无法识别。此外，溶 解曲线定量分析中对峰高、峰宽的数据采集识别也有一定的要求，区分不同样式的溶解曲线较为不易，极易出现偏差，同时，结果判读过程复杂、繁琐且难度高，难于广泛推广应用。
发明内容
本申请提供了一种用于人运动神经元存活基因SMN1拷贝数检测的方法及其引物探针组合。本申请提供的方法及其引物探针组合，在同一反应中同时扩增待测样本的靶基因SMN1和内参基因GAPDH，通过计算qPCR扩增曲线上两者的扩增曲线ΔRn比值C(C＝ΔRn SMN1/ΔRn GAPDH)确定待测个体的SMN1基因拷贝数，根据该比值的范围可有效检测出SMN1基因纯合缺失、杂合缺失和野生型3种基因型。
具体地，本申请基于如下发现：
荧光定量PCR反应中Rn值(Normalized reporter)反映了每个循环的荧光强度，其与基线本底荧光值(Rn-)的差值ΔRn可以表示每个扩增循环中PCR产物的量。PCR反应中的各种组分，如模板浓度、引物浓度、dNTP浓度、Mg 2+浓度等都会对产物扩增效率产生影响。
本申请在PCR扩增体系中同时扩增待测基因SMN1和内参基因GAPDH，二者间存在竞争关系。同时，本申请将反应中的dNTP浓度由正常的200μM降低到80μM以下，以限制待测基因SMN1和内参基因GAPDH的扩增效率。当待测基因SMN1和内参基因GAPDH均为2个拷贝数时，两基因浓度相同(1：1)，反应中两者对限制性试剂dNTP的竞争会达到一个平衡水平，两者的扩增产物也会达到一个平衡水平。此时在qPCR扩增曲线上表现为两者的扩增曲线ΔRn比值C(C＝ΔRn SMN1/ΔRn GAPDH)处在一个稳定的范围内。
当扩增反应中待测基因SMN1的拷贝数减少为1个拷贝，而内参基因GAPDH仍为2个拷贝数时，二者模板浓度比值为1：2。此时待测基因SMN1的模板浓度相对降低，其在扩增体系中的竞争水平降低，因此竞争获得的反应试剂dNTP也相应减少，PCR扩增产物减少，ΔRn值降低。在这种情况下，内参基因GAPDH的竞争能力相对增强，其获得的反应试剂dNTP也会相应增加，PCR扩增产物增加，ΔRn值增加。此时在qPCR扩增曲线上表现为两者的扩增曲线ΔRn比值C(C＝ΔRn SMN1/ΔRn GAPDH)降低，处在一个较低的范围内。
当扩增反应中待测基因SMN1的拷贝数减少为0个拷贝，而内参基因GAPDH仍为2个拷贝数时，此时扩增体系中没有待测基因SMN1的模板，只有内参基因GAPDH的模板。扩增体系中不会有待测基因SMN1的产物，只有内参基因GAPDH的扩增产物，在qPCR扩增曲线上表现为两者的扩增曲线ΔRn比值C(C＝ΔRn SMN1/ΔRn GAPDH)为0。
同理，当待测基因SMN1的拷贝数增加时，qPCR扩增曲线上两者的扩增曲线ΔRn比值C(C＝ΔRn SMN1/ΔRn GAPDH)也会增加。
基于上述申请构思，本申请提供以下技术方案：
第一方面，本申请提供一种检测靶基因拷贝数的方法，所述方法包括：
(1)提取样本DNA；
(2)在同一反应中同时对所述样本DNA中的靶基因和内参基因进行qPCR扩增，分别获得所述靶基因和内参基因的扩增曲线ΔRn值；以及
(3)靶基因拷贝数分析：计算所述靶基因和内参基因的扩增曲线ΔRn值的比值C，根据比值C判断靶基因拷贝数。
优选地，所述靶基因为SMN1，所述内参基因为GAPDH。
优选地，步骤(3)中扩增曲线ΔRn值的比值C的计算公式为：C＝ΔRn 靶 基因/ΔRn 内参基因，进一步优选为：C＝ΔRn SMN1/ΔRn GAPDH，。
优选地，步骤(3)中，判断靶基因拷贝数的标准包括：
SMN1和内参基因GAPDH的扩增曲线ΔRn值的比值C为0.85～1.15，则判断SMN1基因和内参基因GAPDH均为2个拷贝；
SMN1和内参基因GAPDH的扩增曲线ΔRn值的比值C为0.3～0.7，则判断SMN1基因的拷贝数为1个拷贝，内参基因GAPDH为2个拷贝；或
SMN1和内参基因GAPDH的扩增曲线ΔRn值的比值C为-0.1～0.1，则判断SMN1基因的拷贝数为零个拷贝。
优选地，步骤(2)具体包括如下步骤：
将样本DNA、检测靶基因的引物探针组合和检测内参基因的引物探针组合加入检测体系PCR反应液中，混合，得到PCR体系，对PCR体系进行扩增，分别获得所述靶基因和内参基因的扩增曲线ΔRn值。
优选地，所述检测靶基因的引物探针组合包括用于人运动神经元存活基因SMN1拷贝数检测的引物探针组合，所述用于人运动神经元存活基因SMN1拷 贝数检测的引物探针组合包括特异性扩增并检测人运动神经元存活基因SMN1的引物对和探针；
所述特异性扩增人运动神经元存活基因SMN1的引物对包括：
正向引物SEQ ID No:1：TTTATTTTCCTTACAGGGTTTC；
反向引物SEQ ID No:2：GCTGGCAGACTTACTCCTTA；
所述检测人运动神经元存活基因SMN1的探针包括SEQ ID No:3：AGAAGGAAGGTGCTCACATT所示的核苷酸序列。
优选地，所述探针的5’端修饰荧光染料，所述探针的3’端修饰淬灭剂。
优选地，所述探针的3’端还连接了MGB基团。
优选地，所述荧光染料包括FAM。
优选地，所述淬灭剂包括TAMRA。
本申请中，所设计的MGB探针引物利用FAM荧光染料进行5’端修饰，荧光信号强，检测效率最高。
本申请中，SMN1基因的NCBI编码为GenBank:NG_008691.1，所述正向引物及反向引物分别对应于SMN1基因31985～32006和32049～32068碱基，所述MGB探针对应于SMN1基因32021～32040处碱基。
优选地，所述检测内参基因的引物探针组合包括特异性扩增并检测内参基因GAPDH的引物对和探针；
所述特异性扩增内参基因GAPDH的引物对包括：
正向引物SEQ ID No:4：AAGGGCTTCGTATGACTGGG；
反向引物SEQ ID No:5：CTCCCTTGAGCTTCCCTGC；
所述检测内参基因GAPDH的探针包括SEQ ID No:6：TTGGGCAGCCCTGGA所示的核苷酸序列。
优选地，所述探针的5’端修饰荧光染料，所述探针的3’端修饰淬灭剂。
优选地，所述探针的3’端还连接了MGB基团。
优选地，所述荧光染料包括FAM、TET、VIC或HEX中任意一种。
优选地，所述淬灭剂包括BHQ。
本申请中，使用GAPDH基因作为内参基因，扩增效果良好，检测效率高。本申请中设计的正向引物和反向引物的碱基分布随机，引物3’端无连续的G或C碱基聚集，3’端不存在自身互补重叠序列，可避免发夹结构和引物二聚体的产 生。同时，正向引物特意性地设计在SMN1基因和SMN2基因第7外显子中c.840位点的C>T差异碱基处，可避免SMN2基因对SMN1基因扩增产生干扰。
优选地，所述样本DNA的浓度为5～100ng/μL，例如可以是5ng/μL、10ng/μL、20ng/μL、30ng/μL、40ng/μL、50ng/μL、60ng/μL、70ng/μL、80ng/μL、90ng/μL或100ng/μL等。
优选地，所述PCR体系中特异性扩增所述人运动神经元存活基因SMN1的正向引物和反向引物的终浓度各自独立的为0.1～1μM，例如可以是0.1μM、0.3μM、0.5μM、0.7μM、0.9μM或1μM等。
优选地，所述PCR体系中特异性扩增所述内参基因GAPDH的正向引物和反向引物的终浓度各自独立的为0.1～1μM，例如可以是0.1μM、0.3μM、0.5μM、0.7μM、0.9μM或1μM等。
优选地，所述PCR体系中检测人运动神经元存活基因SMN1的探针和检测内参基因GAPDH的探针的终浓度各自独立的为0.1～1μM，例如可以是0.1μM、0.3μM、0.5μM、0.7μM、0.9μM或1μM等。
优选地，所述PCR体系中dNTP的终浓度为80～1.0μM，例如可以是80μM、60μM、40μM、20μM、10μM、5.0μM或1.0μM等。
优选地，所述PCR体系中DNA聚合酶的终浓度为0.02～0.1U/μL，例如可以是0.02U/μL、0.03U/μL、0.05U/μL、0.07U/μL、0.09U/μL或0.1U/μL等。
本申请中，所述PCR扩增的程序包括：
预变性：93～96℃孵育3～7min，孵育的温度例如可以是93℃、94℃、95℃或96℃等，孵育的时间例如可以是3min、4min、5min、6min或7min等；
变性：93～96℃孵育25～35s，孵育的温度例如可以是93℃、94℃、95℃或96℃等，孵育的时间例如可以是25s、27s、29s、30s、31s、33s或35s等；
退火和延伸：
50～55℃孵育85～95s，孵育的温度例如可以是50℃、51℃、52℃、53℃、54℃或55℃等，孵育的时间例如可以是85s、87s、89s、90s、91s、93s或95s等；
70～74℃孵育25～35s，孵育的温度例如可以是70℃、71℃、72℃、73℃或74℃等，孵育的时间例如可以是25s、27s、29s、30s、31s、33s或35s等；
循环38～42次，循环的次数例如可以是38次、39次、40次、41次或42次；
保温：0～4℃保存，例如可以是0℃、1℃、2℃、3℃或4℃等。
优选地，所述方法还包括使用质控试剂进行同步检测的步骤。
本申请的方法中，利用在同一反应中同时扩增待测个体的靶基因SMN1和内参基因GAPDH，并创造性地设计计算拷贝数方法，通过计算qPCR扩增曲线上两者的扩增曲线ΔRn比值C(C＝ΔRn SMN1/ΔRn GAPDH)确定待测样本中的SMN1基因拷贝数，与现有技术中判断方法相比，本申请的判断方法不需要制作qPCR定量标准曲线，操作简单，使用方便，结果准确，有利于广泛推广应用。
优选地，所述使用方法还包括判断SMN1基因的缺失情况的步骤。
优选地，判断SMN1基因的缺失情况的标准包括：
SMN1基因和内参基因GAPDH均为2个拷贝，所述SMN1基因为野生型；
SMN1基因的拷贝数为1个拷贝，内参基因GAPDH为2个拷贝，所述SMN1基因为杂合缺失型；或
SMN1基因的拷贝数为零个拷贝，SMN1无扩增产物，所述SMN1基因为纯合缺失型。
第二方面，本申请提供一种用于人运动神经元存活基因SMN1拷贝数检测的试剂盒，所述用于人运动神经元存活基因SMN1拷贝数检测的试剂盒包括第一方面所述的检测靶基因的引物探针组合、检测内参基因的引物探针组合、反应试剂和质控试剂。
优选地，所述反应试剂包括样本DNA抽提试剂和检测体系PCR反应液。
优选地，所述检测体系PCR反应液包括DNA聚合酶、酶缓冲液、MgCl 2和dNTPs。
优选地，所述质控试剂包括SMN1零拷贝质控品、SMN1单拷贝质控品或SMN1两拷贝质控品。
优选地，所述SMN1零拷贝质控品为含有两拷贝内参基因GAPDH的质粒。
优选地，所述SMN1单拷贝质控品为含有单拷贝SMN1基因和两拷贝内参基因GAPDH的质粒。
优选地，所述SMN1两拷贝质控品为含有两拷贝SMN1基因和两拷贝内参基因GAPDH的质粒。
本申请中，所述用于人运动神经元存活基因SMN1拷贝数检测的试剂盒中设计了检测SMN1的正向引物、反向引物及MGB探针引物，可同时用于荧光 定量PCR及其它PCR技术方法，所述引物的扩增效率高，对质粒标准品及送检样本进行qPCR检测，均可以准确地检测判断患者的SMN1基因型。通过调整正向引物、反向引物、MGB探针的浓度和退火温度等反应条件，可使扩增效率达到最佳。
作为本申请的优选技术方案，所述用于人运动神经元存活基因SMN1拷贝数检测的试剂盒以非疾病诊断和/或治疗为目的的使用方法包括以下步骤：
(1)采集并处理待测DNA样本，将待测DNA样本、用于SMN1、内参基因GAPDH拷贝数检测的引物探针组合加入检测体系PCR反应液中，混合，得到PCR体系；所述PCR体系中所述待测DNA样本的浓度为5～100ng/μL，特异性扩增所述人运动神经元存活基因SMN1的正向引物和反向引物的终浓度各自独立的为0.1～1μM，特异性扩增所述内参基因GAPDH的正向引物和反向引物的终浓度各自独立的为0.1～1μM，检测人运动神经元存活基因SMN1的探针和检测内参基因GAPDH的探针的终浓度各自独立的为0.1～1μM，所述PCR体系中dNTP的终浓度为80～1.0μM，所述PCR体系中DNA聚合酶的终浓度为0.02～0.1U/μL。
在检测时，使用质控试剂进行同步检测。
(2)对PCR体系进行扩增，获得SMN1和GAPDH的扩增曲线。
所述PCR扩增的程序包括：
预变性：93～96℃孵育3～7min；
变性：93～96℃孵育25～35s；
退火和延伸：50～55℃孵育85～95s，70～74℃孵育25～35s，循环38～42次；
保温：0～4℃保存。
(3)利用SMN1和GAPDH的扩增曲线的ΔRn值，进行SMN1基因拷贝数分析，判断SMN1基因的缺失情况，判断SMN1基因的缺失情况的标准包括：
SMN1基因和内参基因GAPDH均为2个拷贝，SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C处于正常水平(0.85-1.15范围)，所述SMN1基因为野生型；
SMN1基因的拷贝数为1个拷贝，内参基因GAPDH为2个拷贝，SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C显著性降低(0.3-0.7范围)，所述SMN1基因为杂合缺失型；或
SMN1基因的拷贝数为零个拷贝，SMN1无扩增产物，SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C为0(-0.1～+0.1范围)，所述SMN1基因为纯合缺失型。
第三方面，本申请提供一种用于人运动神经元存活基因SMN1拷贝数检测的系统，所述用于人运动神经元存活基因SMN1缺失检测的系统包括：
(1)样本制备模块：采集并处理待测DNA样本，将待测DNA样本、用于SMN1、内参基因GAPDH拷贝数检测的引物探针组合加入检测体系PCR反应液中，混合，配制PCR体系；
(2)检测模块：对PCR体系进行扩增检测，获得SMN1和GAPDH的扩增曲线；以及
(3)分析模块：计算SMN1和GAPDH的扩增曲线的ΔRn值，进行SMN1基因拷贝数分析；
其中，用于SMN1、内参基因GAPDH拷贝数检测的引物探针组合，PCR体系，以及SMN1基因拷贝数的判断方法如上述第一和第二方面所述。
本申请所述的数值范围不仅包括上述例举的点值，还包括没有例举出的上述数值范围之间的任意的点值，限于篇幅及出于简明的考虑，本申请不再穷尽列举所述范围包括的具体点值。
相对于现有技术，本申请具有以下有益效果：
(1)本申请中设计的检测方法可有效地检测出SMN1基因的拷贝数，所述检测方法不需要制作定量标准曲线，操作简单，使用方便，根据所述ΔRn比值的范围C值可有效地检测出SMN1基因的拷贝数，并进一步分析纯合缺失、杂合缺失和野生型3种基因型；
(2)本申请中的检测SMN1基因的正向和反向扩增引物，可以高效地扩增出目的基因，具有很高的特异性及精确性，检测效率高，所述MGB探针的特异性好、荧光信号强，检测效率高；
(3)qPCR扩增曲线分析技术具有操作简单方便，结果易于判断，成本低通量大等优点，将极大地提高SMN1基因杂合缺失携带者的检出效率，有效应用于评估SMN1基因检测试剂盒的性能、SMN1基因相关机理研究以及辅助分析检测SMN1基因缺失的中间结果信息等，对于脊髓性肌萎缩症检测领域具有重要意义。本申请中开发的方法不仅能够用于SMN1基因拷贝数检测，同样可 用于其它基因的拷贝数检测。
附图说明
图1为测试例1中SMN1基因两拷贝质控品的检测结果图。
图2为测试例1中SMN1基因单拷贝质控品的检测结果图。
图3为测试例1中SMN1基因零拷贝质控品的检测结果图。
图4为测试例2中野生型样本SMN1基因两拷贝数的检测结果图。
图5为测试例2中杂合缺失样本SMN1基因单拷贝数的检测结果图。
图6为测试例2中纯合缺失样本SMN1基因两拷贝数的检测结果图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
本实施例提供一种用于人运动神经元存活基因SMN1拷贝数检测的引物探针组合，所述用于人运动神经元存活基因SMN1拷贝数检测的引物探针组合包括特异性扩增并检测人运动神经元存活基因SMN1的引物对和探针、特异性扩增并检测内参基因GAPDH的引物对和探针。
特异性扩增所述人运动神经元存活基因SMN1的正向引物的核苷酸序列如SEQ ID No:1所示，反向引物的核苷酸序列如SEQ ID No:2所示；检测所述人运动神经元存活基因SMN1的探针的核苷酸序列如SEQ ID No:3所示，所述探针的5’端修饰FAM，所述探针的3’端修饰TAMRA，所述探针的3’端还连接了MGB基团。
特异性扩增所述内参基因GAPDH的正向引物的核苷酸序列如SEQ ID No:4所示，反向引物的核苷酸序列如SEQ ID No:5所示；检测所述内参基因GAPDH的探针的核苷酸序列如SEQ ID No:6所示，所述探针的5’端修饰HEX，所述探针的3’端修饰BHQ，所述探针的3’端还连接了MGB基团。
SEQ ID No:1：TTTATTTTCCTTACAGGGTTTC。
SEQ ID No:2：GCTGGCAGACTTACTCCTTA。
SEQ ID No:3：AGAAGGAAGGTGCTCACATT。
SEQ ID NO:4：AAGGGCTTCGTATGACTGGG。
SEQ ID NO:5：CTCCCTTGAGCTTCCCTGC。
SEQ ID NO:6：TTGGGCAGCCCTGGA。
实施例2
本实施例提供一种用于人运动神经元存活基因SMN1拷贝数检测的试剂盒，所述用于人运动神经元存活基因SMN1拷贝数检测的试剂盒包括实施例1中所述的用于人运动神经元存活基因SMN1拷贝数检测的引物探针组合、反应试剂和质控试剂。
所述反应试剂包括样本DNA抽提试剂(凯捷公司的提取试剂盒，货号51104)、乙醇和检测体系PCR反应液，所述检测体系PCR反应液包括DNA聚合酶、酶缓冲液、MgCl 2和dNTPs。
所述质控试剂包括SMN1两拷贝质控、SMN1单拷贝质控或SMN1零拷贝质控。所述SMN1两拷贝质控为含有两拷贝SMN1基因和两拷贝内参基因GAPDH的质粒，所述SMN1单拷贝质控为含有单拷贝SMN1基因和两拷贝内参基因GAPDH的质粒，所述SMN1零拷贝质控为含有两拷贝内参基因GAPDH的质粒。上述质控质粒由上海生物工程股份有限公司合成。
测试例1
本测试例使用实施例2中所述的用于人运动神经元存活基因SMN1拷贝数检测的试剂盒测试SMN1零拷贝质控、SMN1单拷贝质控和SMN1两拷贝质控的扩增曲线。
(1)配制PCR体系。
PCR体系如下所示：
Figure PCTCN2022131962-appb-000001
Figure PCTCN2022131962-appb-000002
分别加入SMN1零拷贝质控、SMN1单拷贝质控和SMN1两拷贝质控进行检测。
(2)对PCR体系进行扩增，获得SMN1和GAPDH的扩增曲线。
PCR扩增在ABI7500PCR仪上进行，PCR扩增的程序如下所示：
预变性：94℃孵育5min；
变性：94℃孵育30s；
退火和延伸：55℃孵育90s，72℃孵育30s，循环40次；
保温：4℃保存。
(3)计算SMN1和GAPDH的扩增曲线的ΔRn值，进行SMN1基因拷贝数分析。
检测结果如图1、图2和图3所示，其中SMN1基因两拷贝质控的检测结果图如图1所示，SMN1基因单拷贝质控的检测结果图如图2所示，SMN1基因零拷贝质控的检测结果图如图3所示。
质粒标准品检测结果如表1所示。
表1
样本编号 ΔRn的比值C 质粒标准品种类 qPCR引物检测结果
1 0.96 野生型(两拷贝) 野生型(两拷贝)
2 0.48 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
3 0.02 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
由表1可知，本申请在qPCR基础上，创造性地设计计算拷贝数方法，可快速、准确分析SMN1基因拷贝数，不需要制作qPCR定量标准曲线，操作简单，使用方便。
测试例2
本测试例使用实施例2中所述的用于人运动神经元存活基因SMN1拷贝数检测的试剂盒检测临床标本，评估所述试剂盒的准确性，所述临床标本包括抗 凝血标本24例。
(1)采集并处理待测DNA样本。
抽提全血液样本中的基因组DNA：
用移液枪吸取20μL蛋白酶K溶液置于1.5mL离心管底部；在1.5mL离心管中加入200μL全血液样本；加入200μL Buffer AL，震荡混匀15s，56℃孵育10min；短时离心，使溶液沉降到1.5mL离心管底部；加入200μL无水乙醇，振荡混匀15s，离心；将离心后的上清液全部转入2mL带滤膜的过滤柱子中，盖上管盖，6000g离心1min；在过滤柱中加入500μL Buffer AW1，6000g离心1min；在过滤柱中加入500μL Buffer AW2，20000g离心3min；将过滤柱再放入离心机中离心1min，甩干残留液体；将过滤柱放置于新的1.5mL离心管，加入200μL Buffer AE，6000g离心1min。
配制PCR体系，PCR体系如下所示：
Figure PCTCN2022131962-appb-000003
所述样本DNA模板为从全血液样本中抽提的基因组DNA，所述基因组DNA在PCR体系中的终浓度为50ng/μL。根据检测份数配制PCR反应液。
实验组中每个PCR反应液中加将1μL提取的血液基因组DNA溶液，阳性对照组中直接加1μL阳性对照品(以SMN1两拷贝质控品、SMN1单拷贝质控品和SMN1零拷贝质控品作为阳性对照品)，阴性对照组中直接加1μL ddH 2O。
(2)对PCR体系进行扩增，获得SMN1和GAPDH的扩增曲线。
PCR扩增在ABI7500PCR仪上进行，PCR扩增的程序如下所示：
预变性：94℃孵育5min；
变性：94℃孵育30s；
退火和延伸：55℃孵育90s，72℃孵育30s，循环40次；
保温：4℃保存。
(3)计算SMN1和GAPDH的扩增曲线的ΔRn值，进行SMN1基因拷贝数分析，判断SMN1基因的缺失情况。
判断SMN1基因的缺失情况的标准包括：
SMN1基因和内参基因GAPDH均为2个拷贝，SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C处于0.85-1.15范围，所述SMN1基因为野生型；
SMN1基因的拷贝数为1个拷贝，内参基因GAPDH为2个拷贝，SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C处于0.3-0.7范围，所述SMN1基因为杂合缺失型；或
SMN1基因的拷贝数为零个拷贝，SMN1无扩增产物，SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C为0(-0.1～+0.1范围)，所述SMN1基因为纯合缺失型。
检测结果如图4、图5和图6所示，其中SMN1基因两拷贝的野生型样本检测结果图如图4所示，SMN1基因单拷贝的杂合缺失样本检测结果图如图5所示，SMN1基因零拷贝的纯合缺失样本检测结果图如图6所示。
临床24例样本检测结果如表2所示。
表2
样本编号 ΔRn的比值C SMN1缺失诊断结果 qPCR引物检测结果
1 0.92 野生型(两拷贝) 野生型(两拷贝)
2 1.06 野生型(两拷贝) 野生型(两拷贝)
3 1.12 野生型(两拷贝) 野生型(两拷贝)
4 1.12 野生型(两拷贝) 野生型(两拷贝)
5 0.57 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
6 0.02 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
7 0.70 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
8 0.05 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
9 0.08 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
10 0.44 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
11 0.02 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
12 0.01 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
13 0.01 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
14 0.61 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
15 0.60 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
16 -0.01 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
17 0.60 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
18 0.53 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
19 0.32 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
20 0.30 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
21 0.57 杂合缺失型(单拷贝) 杂合缺失型(单拷贝)
22 -0.00 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
23 -0.00 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
24 -0.00 纯合缺失型(零拷贝) 纯合缺失型(零拷贝)
由表2中可知，上述24例样本的qPCR扩增曲线比值检测结果显示样本中有4例野生型(两拷贝)样本，10例杂合缺失(单拷贝)样本，10例纯合缺失(零拷贝)样本，且与SMN1临床检测拷贝数结果一致，准确率为100％。
综上，本申请中所述用于人运动神经元存活基因SMN1缺失检测的方法及其引物探针组合具有很好的特异性与灵敏度，本申请所提供的用于人运动神经元存活基因SMN1缺失检测的扩增曲线比值检测方法检测准确度高，扩增效果好，使用时操作简单、结果易于判断，检测成本低通量大，极大地提高了SMN1基因杂合缺失携带者的检出效率，对于脊髓性肌萎缩症检测领域具有重要意义。本申请中开发的方法不仅能够用于SMN1基因拷贝数检测，同样可用于其它基因的拷贝数检测。
申请人声明，以上所述仅为本申请的具体实施方式，但本申请的保护范围并不局限于此，所属技术领域的技术人员应该明了，任何属于本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，均落在本申请的保护范围和公开范围之内。
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Claims (12)

  1. 一种检测靶基因拷贝数的方法，其包括：
    (1)提取样本DNA；
    (2)在同一反应中同时对所述样本DNA中的靶基因和内参基因进行qPCR扩增，分别获得所述靶基因和内参基因的扩增曲线ΔRn值；以及
    (3)靶基因拷贝数分析：计算所述靶基因和内参基因的扩增曲线ΔRn值的比值C，根据比值C判断靶基因拷贝数。
  2. 根据权利要求1所述的方法，其中，所述靶基因为SMN1，所述内参基因为GAPDH。
  3. 根据权利要求1或2所述的方法，其中，步骤(3)中，扩增曲线ΔRn值的比值C的计算公式为：C＝ΔRn 靶基因/ΔRn 内参基因，进一步优选为：C＝ΔRn SMN1/ΔRn GAPDH，。
  4. 根据权利要求1-3任一项所述的方法，其中，步骤(3)中，判断靶基因拷贝数的标准包括：
    SMN1和内参基因GAPDH的扩增曲线ΔRn值的比值C为0.85～1.15，则判断SMN1基因和内参基因GAPDH均为2个拷贝；
    SMN1和内参基因GAPDH的扩增曲线ΔRn值的比值C为0.3～0.7，则判断SMN1基因的拷贝数为1个拷贝，内参基因GAPDH为2个拷贝；或
    SMN1和内参基因GAPDH的扩增曲线ΔRn值的比值C为-0.1～0.1，则判断SMN1基因的拷贝数为零个拷贝。
  5. 根据权利要求2-4任一项所述的方法，其中，步骤(2)具体包括如下步骤：将样本DNA、检测靶基因的引物探针组合和检测内参基因的引物探针组合加入检测体系PCR反应液中，混合，得到PCR体系，对PCR体系进行扩增，分别获得所述靶基因和内参基因的扩增曲线ΔRn值；
    优选地，所述检测靶基因的引物探针组合包括用于SMN1拷贝数检测的引物探针组合，所述用于SMN1拷贝数检测的引物探针组合包括特异性扩增并检测SMN1的引物对和探针；
    优选地，所述特异性扩增SMN1的引物对包括，
    正向引物SEQ ID No:1：TTTATTTTCCTTACAGGGTTTC；
    反向引物SEQ ID No:2：GCTGGCAGACTTACTCCTTA；
    优选地，所述检测SMN1的探针包括SEQ ID No:3： AGAAGGAAGGTGCTCACATT所示的核苷酸序列；
    优选地，所述检测内参基因的引物探针组合包括特异性扩增并检测内参基因GAPDH的引物对和探针；
    优选地，所述特异性扩增内参基因GAPDH的引物对包括，
    正向引物SEQ ID No:4：AAGGGCTTCGTATGACTGGG；
    反向引物SEQ ID No:5：CTCCCTTGAGCTTCCCTGC；
    优选地，所述检测内参基因GAPDH的探针包括SEQ ID No:6：TTGGGCAGCCCTGGA所示的核苷酸序列；
    优选地，所述探针的5’端修饰荧光染料，所述探针的3’端修饰淬灭剂；
    优选地，所述探针的3’端还连接了MGB基团。
  6. 根据权利要求5所述的方法，其中，所述样本DNA的浓度为5～100ng/μL；
    优选地，所述PCR体系中特异性扩增SMN1的正向引物和反向引物的终浓度各自独立的为0.1～1μM；
    优选地，所述PCR体系中特异性扩增所述内参基因GAPDH的正向引物和反向引物的终浓度各自独立的为0.1～1μM；
    优选地，所述PCR体系中检测SMN1的探针和检测内参基因GAPDH的探针的终浓度各自独立的为0.1～1μM；
    优选地，所述PCR体系中dNTP的终浓度为80～1.0μM；
    优选地，所述PCR体系中DNA聚合酶的终浓度为0.02～0.1U/μL。
  7. 根据权利要求2-6任一项所述的方法，其中，所述方法还包括使用质控试剂进行同步检测的步骤；
    优选地，所述使用方法还包括判断SMN1基因的缺失情况的步骤；
    所述判断SMN1基因的缺失情况的标准包括：
    SMN1基因和内参基因GAPDH均为2个拷贝，所述SMN1基因为野生型；
    SMN1基因的拷贝数为1个拷贝，内参基因GAPDH为2个拷贝，所述SMN1基因为杂合缺失型；或
    SMN1基因的拷贝数为零个拷贝，SMN1无扩增产物，所述SMN1基因为纯合缺失型。
  8. 一种用于靶基因拷贝数检测的试剂盒，其包括权利要求5-7中任一项所述的检测靶基因的引物探针组合和检测内参基因的引物探针组合，以及反应试 剂和质控试剂。
  9. 根据权利要求8所述的试剂盒，其中，所述反应试剂包括样本DNA抽提试剂和检测体系PCR反应液；
    优选地，所述检测体系PCR反应液包括DNA聚合酶、酶缓冲液、MgCl 2和dNTPs；
    优选地，所述质控试剂包括SMN1零拷贝质控品、SMN1单拷贝质控品或SMN1两拷贝质控品；
    优选地，所述SMN1零拷贝质控品为含有两拷贝内参基因GAPDH的质粒；
    优选地，所述SMN1单拷贝质控品为含有单拷贝SMN1基因和两拷贝内参基因GAPDH的质粒；
    优选地，所述SMN1两拷贝质控品为含有两拷贝SMN1基因和两拷贝内参基因GAPDH的质粒。
  10. 根据权利要求8或9所述的试剂盒以非疾病诊断和/或治疗为目的的使用方法，包括以下步骤：
    (1)采集并处理样本DNA，将样本DNA、用于SMN1、内参基因GAPDH拷贝数检测的引物探针组合加入检测体系PCR反应液中，混合，得到PCR体系；所述PCR体系中所述样本DNA的浓度为5～100ng/μL，特异性扩增SMN1的正向引物和反向引物的终浓度各自独立的为0.1～1μM，特异性扩增所述内参基因GAPDH的正向引物和反向引物的终浓度各自独立的为0.1～1μM，检测SMN1的探针和检测内参基因GAPDH的探针的终浓度各自独立的为0.1～1μM，所述PCR体系中dNTP的终浓度为80～1.0μM，所述PCR体系中DNA聚合酶的终浓度为0.02～0.1U/μL；
    使用质控试剂进行同步检测；
    (2)对PCR体系进行扩增，获得SMN1和GAPDH的扩增曲线；
    所述PCR扩增的程序包括：
    预变性：93～96℃孵育3～7min；
    变性：93～96℃孵育25～35s；
    退火和延伸：50～55℃孵育85～95s，70～74℃孵育25～35s，循环38～42次；
    保温：0～4℃保存；以及
    (3)利用SMN1和GAPDH的扩增曲线的ΔRn值，进行SMN1基因拷贝 数分析，判断SMN1基因的缺失情况。
  11. 根据权利要求10所述的以非疾病诊断和/或治疗为目的的使用方法，其中，所述判断SMN1基因的缺失情况的标准包括：
    SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C处于正常水平，在0.85-1.15范围，所述SMN1基因为野生型；
    SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C显著性降低，在0.3-0.7范围，所述SMN1基因为杂合缺失型；或
    SMN1和内参基因GAPDH的扩增曲线ΔRn间的比值C为0，在-0.1～+0.1范围，所述SMN1基因为纯合缺失型。
  12. 一种用于人运动神经元存活基因SMN1拷贝数检测的系统，其包括：
    (1)样本制备模块：采集并处理样本DNA，将样本DNA、用于SMN1、内参基因GAPDH拷贝数检测的引物探针组合加入检测体系PCR反应液中，混合，配制PCR体系；
    (2)检测模块：对PCR体系进行扩增检测，获得SMN1和GAPDH的扩增曲线；以及
    (3)分析模块：计算SMN1和GAPDH的扩增曲线的ΔRn值的比值C，进行SMN1基因拷贝数分析。
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