WO2021047175A1 - 基因拷贝数定量分析 - Google Patents

基因拷贝数定量分析 Download PDF

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WO2021047175A1
WO2021047175A1 PCT/CN2020/086108 CN2020086108W WO2021047175A1 WO 2021047175 A1 WO2021047175 A1 WO 2021047175A1 CN 2020086108 W CN2020086108 W CN 2020086108W WO 2021047175 A1 WO2021047175 A1 WO 2021047175A1
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gene
seq
competitor
target gene
internal reference
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丁春明
金胜男
金伟江
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浙江中创生物医药有限公司
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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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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to the field of gene detection, and more specifically to the quantitative analysis of gene copy number.
  • Gene copy number variation is a structural variation that is widespread in the human genome. Gene copy number abnormalities were first discovered in the genome of patients, such as patients with genetic diseases and tumor patients; however, subsequent studies have found that gene copy number abnormalities are also widespread in normal individuals. Although the frequency of copy number abnormality is relatively low, the size of the fragments with abnormal copy number ranges from 1 kb to several Mb, and the length of the gene sequence involved is relatively long, which is closely related to human health and disease. Studies have shown that many gene copy number abnormalities are closely related to the onset and disease phenotype of the disease. The detection of abnormal gene copy number is very important for the diagnosis and treatment of diseases, especially for certain genetic diseases (such as spinal muscular atrophy and ⁇ -thalassemia).
  • Spinal muscular atrophy is one of the main genetic diseases that cause death in infants and young children. It is an autosomal recessive genetic neurodegenerative disease characterized by spinal motor neuron degeneration, skeletal muscle atrophy and general weakness. The incidence of spinal muscular atrophy is 1/6000-1/10000, and the frequency of carriers in different populations worldwide is 1/40-1/50.
  • SMN gene survival motor neuron gene
  • 5qSMA spinal cord motor neuron degeneration
  • SMN2 there is a highly homologous gene SMN2 near the region where the SMN1 gene is located (the similarity is as high as 99%, which is extremely difficult to distinguish).
  • exon 7 840C>T, 8 Exon 1239G>A there are more than ten differences in the intron region. Due to the difference of 840C>T on exon 7 of SMN1 and SMN2, SMN2 has exon skipping during transcription to generate mRNA and cannot be spliced correctly. Only about 10% of SMN2 transcripts are full-length and can be translated successfully. Functional SMN protein.
  • SMN2 copy number is related to the phenotype of patients with spinal muscular atrophy
  • nusinersen for the treatment of spinal muscular atrophy is based on correcting the correct splicing of SMN2 mRNA to generate more functional SMN protein Play a therapeutic role. Therefore, the main contents of SMN gene detection include: SMN1 gene deletion detection, SMN2 gene copy number detection, and SMN1 gene pathogenic point mutation detection.
  • Alpha-thalassemia is caused by mutations in the alpha globin gene (HBA gene), which leads to reduced or inability to synthesize the alpha peptide chain, which leads to changes in the composition of hemoglobin, which in turn leads to chronic hemolysis and anemia.
  • HBA genes include two homologous genes HBA1 and HBA2, two copies of each in normal people.
  • HBA1 gene and HBA2 gene HBA gene defects include HBA1 and HBA2 gene deletion or pathogenic point mutations. About 80-90% of people with HBA gene defects are caused by partial or complete deletion of HBA1 or HBA2, and 10%-20% of people with HBA gene defects are caused by pathogenic point mutations in HBA1 or HBA2 genes.
  • HBA gene defects and the severity of the disease are related to the number of copies of the HBA gene involved.
  • this makes it difficult to quantify the copy number of HBA1 and HBA2.
  • the methods commonly used for gene copy number detection mainly include: multiplex ligation-dependent probe amplification (MLPA), real-time fluorescent quantitative PCR (qPCR), etc.
  • Each set of probes includes left probe oligo (LPO) and right probe oligo (RPO). After the probe is combined with the target gene, the left and right probes of the same group are connected through the action of ligase.
  • the sequence of each set of probes contains the same primer sequence (forward primer sequence & reverse primer sequence) and a tag sequence (stuffer sequence) used to distinguish different products (bases contained in the tag sequence) except for the hybridization sequence that binds to the target gene.
  • the number is different, so that the final corresponding ligation products of different sets of probes have different lengths).
  • all ligation products can be amplified by the same pair of PCR primers, and then capillary electrophoresis is used to distinguish ligation products of different lengths to distinguish product information at different target sites.
  • the gene copy number is analyzed through two-step calibration results: 1In-sample calibration: compare each target gene product with the internal reference gene product to obtain their respective ratios (target gene/internal reference gene); 2Sample calibration: each sample to be tested The ratio of the target gene/internal reference gene and the ratio of the target gene/internal reference gene of the normal control sample (the copy number of each gene to be tested is 2) obtains the corrected ratio, which is used for the final evaluation of the absolute copy number of each gene.
  • the detection process of multiple ligation probe amplification technology is cumbersome, time-consuming, and high detection cost.
  • the probe design of this technology is more difficult, and the result analysis is more complicated. It is limited in the multiple detection of SMN1 pathogenic point mutations, and it is difficult to design a specific multi-point mutation detection program.
  • the ligation reaction may fail, and there will be no subsequent amplification products, resulting in false negative results.
  • Real-time fluorescent quantitative PCR technology involves the design of PCR amplification primers and specific probes on the target gene sequence; at the same time, PCR amplification primers and specific probes are used as internal controls on the gene sequence without copy number variation.
  • different fluorescent signals are used to label the target gene and the internal reference gene respectively.
  • the fluorescent signal used as a marker is collected in real time.
  • the control sample can be the artificially synthesized sequence of the target gene and the artificially synthesized sequence of the internal reference gene mixed in a certain proportion).
  • the single-tube reaction throughput of this detection scheme is low, and it is difficult to detect multiple copies of different gene copy number variations.
  • the sensitivity and specificity of this scheme are limited by the design of different gene detection, and the sensitivity and specificity of different gene detection designs are different. When the sensitivity is not good, although patients with homozygous deletion can be accurately detected (the copy number is 0), it is difficult to accurately quantify the copy number (more than 1/2/3/4/4 copies).
  • the art needs a method that can detect gene copy number more accurately and quickly.
  • the matrix-assisted laser desorption ionization time of flight mass spectrometry (Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry) technology platform combines single-base extension reaction, real-competitive PCR technology detection, and subsequent multi-step calibration completes the gene copy number The accurate quantification.
  • This method includes laboratory methods and data analysis methods, which can accurately detect gene copy number variations (including homozygous/heterozygous deletions and duplications) in the human genome.
  • this technology can also design detection schemes based on the different bases of these sequences.
  • the single base extension reaction can distinguish the different bases and complete the quantification of homologous sequences.
  • the application of the invention can complete SMN1 gene deletion detection, SMN2 gene copy number detection, and copy number quantification of HBA1 and HBA2 in a single tube reaction.
  • the nucleic acid flight mass spectrometry platform has been automated, so the present invention is convenient for clinical promotion and application, and can accurately and comprehensively analyze gene copy number changes, and has high application value.
  • the present invention includes the following embodiments:
  • the present invention relates to a method for detecting the copy number of a target gene, including:
  • the competitor of the gene is at least 1 nucleotide different from the target gene and has at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% from the target gene %, more preferably at least 95%, most preferably at least 99% sequence identity, as a competitive amplification template with the target gene to be amplified simultaneously in the PCR amplification reaction, and the extension primer of the target gene is in the extension reaction
  • the target gene and the amplification product of the competitor of the target gene can be respectively extended in the target gene; the competitor of the internal reference gene has at least one nucleotide sequence difference with the internal reference gene and has at least 50% difference from the internal reference gene.
  • amplification template is amplified simultaneously in the PCR amplification reaction, and the extension primer of the internal reference gene can respectively extend the amplification product of the internal reference gene and the competitor of the internal reference gene in the extension reaction;
  • step b) Add the amplification primers described in step a) to the test DNA sample obtained from the subject suspected of having the copy number variation of the target gene and the normal control DNA sample obtained from normal people, and perform PCR amplification to obtain the target PCR amplification products of genes, internal reference genes and their respective competitors;
  • step c) adding the extension primer described in step a) to the PCR amplification product, and performing an extension reaction to obtain the extension product of the target gene, the internal reference gene and their respective competitors;
  • Example calibration Calculate the calibration ratio of the target gene in the sample to be tested, that is, the ratio of internal reference gene/competitor of internal reference gene, and obtain the ratio of target gene/target gene competitor; use target gene/target gene competitor Divide the ratio of, by the ratio for calibration, to obtain the ratio of the target gene after the initial calibration, that is: the ratio of the target gene to the target gene competitor/the ratio of the internal reference gene to the internal reference gene competitor;
  • step i) Calibration between samples: According to the method of step i), calculate the ratio of the target gene in the normal control sample after the initial calibration; compare the ratio of the target gene in the sample to be tested obtained in step i) with the ratio of the target gene in the normal control sample The ratio of the target gene after the initial calibration is compared to obtain the final calibration ratio of the target gene, that is, the ratio of the target gene after the initial calibration is the sample to be tested /the target gene is the normal reference sample after the initial calibration;
  • the present invention relates to a kit for detecting the copy number of a target gene, which contains amplification and extension primers for the target gene, a competitor for the target gene, and amplification and extension of an internal reference gene without copy number variation A primer, and a competitor of the internal reference gene, wherein the competitor of the target gene is at least 1 nucleotide different from the target gene and has at least 50%, preferably at least 60%, and more Preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably at least 99% sequence identity, the competitor of the internal reference gene and the internal reference gene have at least 1 core
  • the difference between the glycidyl acid and the internal reference gene is at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, most preferably at least 99% Sequence identity.
  • the DNA sample is selected from body fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, feces and Tissue sample.
  • nucleotide differences there is a difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides between the competitor of the target gene and the target gene, and the internal reference gene.
  • the target gene is SMN1, SMN2, NAIP, HBA1, HBA2 and/or HBQ gene.
  • the amplification and extension primers of the target gene are selected from i) the amplification and extension primers of SMN17 exon and SMN2 exon 7, which are as SEQ ID NO: 1-2 and SEQ ID, respectively NO: 6; ii) SMN1 exon 8 and SMN2 exon 8 amplification and extension primers, which are shown in SEQ ID NO: 3-4 and SEQ ID NO: 5, respectively; iii) NAIP gene 5
  • the amplification and extension primers of exon number are shown in SEQ ID NO: 13-14 and SEQ ID NO: 15 respectively;
  • the amplification and extension primers of HBA1 gene are shown in SEQ ID NO: 17-18, respectively And SEQ ID NO: 24; v) HBA2 gene amplification and extension primers, which are shown in SEQ ID NO: 19-20 and SEQ ID NO: 26, respectively; and vi) HBQ gene amplification and extension primers , which are shown in SEQ ID NO: 21-22 and SEQ ID
  • the competitor of the target gene is selected from i) competitors of SMN17 exon and SMN2 exon 7, which are shown in SEQ ID NO: 7; ii) SMN1 exon 8 and SMN2
  • the competitor of exon 8 is shown in SEQ ID NO: 8;
  • iii) the competitor of exon 5 of the NAIP gene is shown in SEQ ID NO: 16;
  • iv) the competitor of HBA1 gene is shown in SEQ ID NO: 23, v) HBA2 gene competitor, which is shown in SEQ ID NO: 25, and vi) HBQ gene competitor, which is shown in SEQ ID NO: 27.
  • the internal reference gene is the NFATC3 gene
  • the amplification and extension primers of the internal reference gene are shown in SEQ ID NO: 9-10 and 11, respectively
  • the competitor of the internal reference gene is shown in SEQ ID NO: 12 shown.
  • the present invention relates to primers, which are selected from SEQ ID NO: 1-6, SEQ ID NO: 9-11, SEQ ID NO: 13-15, SEQ ID NO: 17-22, SEQ ID NO: 24 , SEQ ID NO: 26 and SEQ ID NO: 28 are shown in the sequence.
  • the present invention relates to a polynucleotide, which is shown in a sequence selected from SEQ ID NO: 7, 8, 12, 16, 23, 25, and 27.
  • the invention can accurately detect gene copy number variation, and can also be applied to the accurate copy number detection of highly homologous genes.
  • the present invention can perform gene copy number detection on the same platform and provide a more comprehensive analysis of disease and health-related genes.
  • the present invention can be directly quantified by nanodrop after DNA extraction, even without DNA quantification steps.
  • the ideal sample load range of the sample to be tested can be 10-80ng, and the normal reference sample can choose any amount in this interval, such as 10ng, 20ng or 40ng can be used for subsequent calibration to further streamline the operation and reduce the cost of testing.
  • the nucleic acid flight mass spectrometry platform used in the present invention has been automated, has a simple process, and takes relatively little time (about 8 hours), and is convenient for clinical promotion and application. Many disease-related copy number variation detection schemes can be designed through the present invention, and they can be applied in clinical practice.
  • Figure 1 is a schematic diagram of the quantitative detection scheme of SMN1, SMN2, NAIP gene copy number and expected results: Taking the design on exon 7 of SMN1 gene as an example, the difference bases between exon 7 of SMN1 gene and exon 8 of SMN2 gene Design PCR amplification primers upstream and downstream to amplify the sequence.
  • a competitor is introduced as a competitive template in addition to the gDNA template, and the sequence of the competitor is the same as the sequence of the gDNA amplicon except for the difference bases.
  • the extension primers were designed. Different bases have different molecular weights.
  • Figure 2 shows the detection results of SMN1 gene exon 7 and SMN2 gene exon 8 of the SMN1, SMN2, NAIP gene copy number quantitative detection program in clinical samples (normal, carrier, patient). To achieve the expected detection efficiency, it can accurately distinguish and diagnose the three groups of normal, carrier, and patient groups.
  • Figure 3 shows the SMN1 gene, SMN2 gene, NAIP gene copy number quantification scheme copy number correction test results: At present, most other copy number quantification detection schemes require more accurate and consistent template loading, and the design scheme and correction scheme of this scheme are There is a large flexibility space for the requirement of sample amount on the template. Fifteen repetitions were made with 10ng, 20ng, 40ng, and 80ng loading volume respectively.
  • Figure 3A the calculation result of the copy number of each reaction when using 4 random 10ng loading reactions as the normal reference sample calibration
  • Figure 3B the calculation result of the copy number of each reaction using 4 random 20ng loading reactions as the normal reference sample calibration
  • Figure 3C shows the calculation results of the copy number of each reaction when using 4 random 40ng loading reactions as the normal reference sample for calibration
  • Figure 3D shows the calculation result of the copy number of each response when using 4 random 80ng loading reactions as the normal reference sample for calibration.
  • the results show that when the 20ng loading reaction is used as a normal reference sample for calibration, the 10ng, 20ng, and 40ng loading reactions can all obtain more accurate copy number results.
  • Figures 4A-4D show the results of the storage stability test of the competitor mixture: the competitive mixture was tested under different storage conditions with a copy number quantitative detection scheme.
  • Figure 4A the copy number quantification result of the competition mixture under the storage condition of -20°C with carrier DNA added
  • Figure 4B the copy number quantification result of the competition mixture under the storage condition of 4°C with carrier DNA added
  • Figure 4C The copy number quantification results of the competition mixture stored at -20°C without the addition of carrier DNA
  • Figure 4D the copy number quantification results of the competition mixture stored at 4°C without the addition of carrier DNA.
  • the results show that the competitor mixture has good stability under the above four storage conditions, and when used in a copy number quantitative detection program, the copy number can be quantified stably and accurately.
  • Figures 5A-5C show the results of repeated freezing and thawing tests of the competitor: performing multiple freeze-thaw tests on the competitor mixture to simulate multiple freeze-thaw cycles in actual use, and test its stability after repeated freeze-thaw cycles.
  • the competitor mixture was subjected to 20 repeated freezing and thawing cycles, and the competitor mixture with different freezing and thawing times was used to test the copy number quantitative detection scheme.
  • Figure 5C using different freeze-thaw times without carrier DNA-added competitor mixtures for copy number quantitative detection result. The results show that the competitor mixture with and without carrier DNA is stable in repeated freezing and thawing, and can accurately detect the copy number quantitatively.
  • Figure 6 shows a schematic diagram of the design of the HBA gene copy number detection scheme: for the three common deletion types of HBA genes, three highly conserved regions near the HBA1 and HBA2 genes were selected as targets for the design of the detection scheme. As shown, ⁇ 4.2 deletion upstream of the target gene deletions only HBA2 HBA2, ⁇ 3.7 HBA1 deletion upstream of the gene deletions only targets H BA1, - SEA deletion upstream of the target gene while deletion HBA2 HBA2, upstream of a target gene HBA1 Click HBA1 , and the upstream target of HBQ gene HBAQ .
  • the present invention designs PCR amplification primers for exon 7 and exon 8 of SMN gene, and simultaneously amplifies SMN1 and SMN2 exon 7/8 regions (the amplified region includes SMN1 and SMN2 exon 7). Differential sites, namely exon 7 C840T, exon 8 G1239A). Extension primers were designed for the two different sites of SMN1 and SMN2. Therefore, SMN1 and SMN2 can be distinguished in the subsequent single-base extension reaction. SMN1/SMN2 can be obtained through the nucleic acid flight mass spectrometry platform to complete the SMN1 and SMN2 Relative quantitative.
  • PCR primers The names and sequences of PCR primers are as follows:
  • SMN_c.C840T_F AACTTCCTTTATTTTCCTTAC (SEQ ID NO: 1)
  • SMN_c.C840T_R TAATGCTGGCAGACTTACTCC (SEQ ID NO: 2)
  • SMN_c.G1239A_F GTGAAATATTTTACTGGACTC (SEQ ID NO: 3)
  • SMN_c.G1239A_R TTTTCTCAACTGCCTCACCAC (SEQ ID NO: 4)
  • extension primers are named and sequenced as follows:
  • SMN_c.G1239A_U CCTCCCACCCCCACC (SEQ ID NO: 5)
  • SMN_c.C840T_U TTTATTTTCCTTACAGGGTTT (SEQ ID NO: 6)
  • step B Design at least two competitor sequences, corresponding to the target sequence amplified by the two pairs of PCR primers designed in step A, respectively, introduce sequence variation at the following positions (C840T>G, G1239A>C) (underlined bases) Is the introduced variant base).
  • SMN1 and SMN2 select the gene sequence without copy number variation in the human genome as an internal control.
  • This sequence is a sequence located on exon 2 of human chromosome 16 NFATC3 gene.
  • DECIPHER v9.30 database this database contains genomic data of 30405 people) and has no copy number in its database population.
  • the selected internal reference sequence PCR amplification primer and extension primer sequence are as follows:
  • QC_g.G3C_F ATATAGCCCATTAGGTGGTCC (SEQ ID NO: 9)
  • QC_g.G3C_U TTGAATACTTGGGCACT (SEQ ID NO: 11)
  • step B introduces a base design competitor that is different from the reference sequence to the test site of the amplicon, its name and sequence are as follows:
  • the extension products corresponding to the genomic DNA template and the competitor template can be distinguished by the nucleic acid flight mass spectrometry platform, and the QC/competitor is obtained for subsequent calibration, which we call the internal reference calibration ratio.
  • PCR amplification primers, extension primers and competitor sequences are as follows:
  • NAIP-E5_c.G5C_F GGAACCATTTGGCATGTTCCT (SEQ ID NO: 13)
  • NAIP-E5_c.G5C_R ACAGTGTTTTTCCTGTGGTGG (SEQ ID NO: 14)
  • NAIP-E5_c.G5C_U TGGGAAGAAGGAGATGAT (SEQ ID NO: 15)
  • E. In-sample calibration (1). Through the design of the corresponding competitor in step AD, we can obtain the ratio of the target gene and the competitor in each design, including SMN1 E7/competitor, SMN2 E7/competitor, SMN1 E8 /Competitor, SMN2 E8/Competitor, NAIP E5/Competitor and internal reference DNA sequence correction ratio QC/Competitor.
  • the competitor added in each reaction is a fixed amount, so the above target gene ratio and the calibration ratio can reflect the absolute amount of the target gene and the internal reference gene, and this ratio allows us to reduce the difference due to the sample itself during subsequent calibration. The influence of PCR amplification reaction makes quantification more accurate. (2).
  • the ratio of each target gene obtained in the sample with the calibration ratio of the internal reference DNA sequence (stable and normal copy number)
  • the ratio obtained at this time after the initial calibration avoids the influence caused by the difference in the amplification efficiency of the PCR reaction between the target sequence and the internal reference DNA sequence.
  • the design of MLPA there is no design of similar competitors, so that it can only obtain the target gene/internal reference gene when calibrating in the sample. If you need to reduce the impact of sample differences or differences in amplification efficiency, you need to perform data More cumbersome correction.)
  • the sample here includes the unknown sample to be tested and 3 to 4 reference samples with known copy numbers for further correction.
  • step AF we obtain the ratio of each target gene after the initial calibration of the sample to be tested and one or more normal reference samples. Compare the ratio after initial calibration in the sample to be tested with the ratio after initial calibration in the normal reference sample to obtain the final calibration ratio of each target gene, such as [SMN1 E7 ratio after initial calibration sample to be tested /SMN1 E7 ratio after primary calibration Normal reference sample ].
  • the copy number of the target gene in the normal reference sample is 2
  • SMN1 E7 copy number [SMN1 E7 after primary calibration
  • the influence caused by the difference in the amount of DNA loaded has been greatly reduced in step F.
  • the sample load of the sample to be tested is within a certain interval, and at the same time, select the normal reference sample data with a suitable sample amount within the interval for correction to obtain the target gene copy numbers of these unknown samples with different sample sizes.
  • the rough nanodrop quantification of the sample to be tested does not even need to be quantified in advance. It can be obtained by setting multiple normal samples with different loading volumes for calibration. Measure the copy number of the target gene in the sample. (In MLPA, in the same batch of experiments, it is necessary to ensure that the sample amount of each sample to be tested is consistent with the normal reference sample before the experiment can be performed.)
  • This protocol is designed to detect the difference bases between exon 7 and exon 8 of SMN1 gene and SMN2 gene ( Figure 1). This protocol can accurately distinguish people with different SMN1 copy numbers and different SMN2 copy numbers ( figure 2).
  • This copy number quantitative detection program can be applied to more single-gene diseases with deletion defects with clear mechanisms.
  • Alpha-thalassemia is caused by HBA gene defects, 90% of HBA gene defects are deletions, so this copy number quantitative detection program can also be applied to the detection of HBA gene deletions. Therefore, we have added a copy number detection program of HBA gene on the basis of the perfect SMN gene detection program.
  • the SMN gene copy number quantitative detection part is as described in the above implementation plan, and the additional HBA gene copy number detection plan is as follows:
  • About 50% of the carriers of the HBA gene defect are SEA-deficient ⁇ /-- SEA
  • about 15% of the carriers of the HBA gene defect are the 3.7 deletion ( ⁇ / ⁇ 3.7 )
  • about 15% are carriers of the HBA gene defect.
  • It is 4.2 deletion type ( ⁇ / ⁇ 4.2 ).
  • about 10% of patients have SEA deletion, 3.7 deletion, and 4.2 deletion complex gene defects (such as: - SEA / ⁇ 4.2 , - SEA / ⁇ 3.7 etc.).
  • this program designs the detection program on the specific sequences upstream of the HBA2 gene, upstream of the HBA1 gene, and upstream of the HBQ gene.
  • the detection scheme is designed on the specific sequences upstream of the HBA2 gene, the upstream of the HBA1 gene, and the upstream of the HBQ gene. Therefore, the PCR amplification of these three regions
  • the primer design is as follows.
  • HBA1_F ACGTTGGATGTCAGCACCCTTCAGCCTGCTC (SEQ ID NO: 17)
  • HBA1_R ACGTTGGATGTTCTCTGCCCAAGGCAGCTTA (SEQ ID NO: 18)
  • HBA2_F ACGTTGGATGGAGACACTTCACTGAGAATAGG (SEQ ID NO: 19)
  • HBA2_R ACGTTGGATGATCTACAACTACTGCCACAGG (SEQ ID NO: 20)
  • HBQ_F ACGTTGGATGTGCCATAGGTGTTTACCAAGG (SEQ ID NO: 21)
  • HBQ_R ACGTTGGATGAGCTGGTAGCCATAAAGCCCTG (SEQ ID NO: 22)
  • HBA1_U AGGCAGCTTACCCTGG (SEQ ID NO: 24)
  • HBA2_U tCTCTCTTTTTGGACAAAAATAC (SEQ ID NO: 26)
  • HBQ_U agtAATATCTTTTATTCCCTGAGC (SEQ ID NO: 28)
  • the ratio of each target gene obtained in the sample with the calibration ratio of the internal reference DNA sequence (stable and normal copy number)
  • the ratio obtained at this time after the initial calibration avoids the influence caused by the difference in the amplification efficiency of the PCR reaction between the target sequence and the internal reference DNA sequence.
  • the design of MLPA there is no design of similar competitors, so that it can only obtain the target gene/internal reference gene when calibrating in the sample. If you need to reduce the impact of sample differences or differences in amplification efficiency, you need to perform data More cumbersome correction.)
  • the sample here includes the unknown sample to be tested and 3 to 4 reference samples with known copy numbers for further correction.
  • steps A-E Calibration between samples: In steps A-E, we obtain the ratio of each target gene after the initial calibration obtained by analyzing the unknown sample to be tested and one or more normal reference samples, which is proportional to the copy number of each target gene in the sample. Therefore, it is known that the copy number of each target gene in the normal sample is 2, and the final correction ratio of each target gene is obtained by comparing the ratio after the initial calibration in the unknown sample with the ratio after the initial calibration in the normal reference sample, such as [SMN1 E7 Samples to be tested for ratios after initial calibration/SMN1 E7 Reference samples for normal ratios after initial calibration].
  • SMN1 E7 copy number [SMN1 E7 after initial calibration Sample of ratio to be tested/SMN1 E7 reference sample of normal ratio after initial calibration]*2.
  • the loading amount of the unknown sample to be tested is within a certain interval, and at the same time, select the normal reference sample data with a suitable loading amount in the interval to calibrate to obtain the target gene copy of the unknown sample to be tested with different loading amounts. number.
  • the sample type and the method of DNA extraction are determined and stable, the rough nanodrop quantification of the unknown sample to be tested does not even need to be quantified in advance. It can be used as a calibration by setting multiple normal samples with different loading amounts. Obtain the copy number of the target gene of the unknown sample to be tested. (In MLPA, in the same batch of experiments, it is necessary to ensure that each unknown sample to be tested is consistent with the normal reference sample before the experiment can be performed.)
  • Example 1 Using the scheme of the present invention and the MLPA scheme for simultaneous copy number quantitative test and comparison
  • Sample detection add the sample DNA to be tested or the reference sample DNA to the reaction wells in sequence, with a sample volume of 2 ⁇ L; add the competitor mixture to the reaction wells in sequence as a competitive template, with a sample volume of 2 ⁇ L.
  • the SAP reaction system configuration is as follows:
  • the SAP reaction program settings are as follows:
  • the extension reaction program settings are as follows:
  • step D Process the data obtained in step D to obtain the ratio of the target gene and the competitor in the sample to be tested, including SMN1 E7/competitor, SMN2 E7/competitor, SMN1 E8/competitor, SMN2 E8/ Competitor, NAIP E5/competitor and internal reference DNA sequence calibration ratio QC/competitor.
  • step b Calibration between samples: same as step a, obtain the ratio of each target gene in the normal reference sample after initial calibration, and compare the ratio of each target gene in the sample to be tested obtained in step a with the corresponding target gene in the normal reference sample The ratios after the initial calibration are compared to obtain the final calibration ratios of each target gene, such as [SMN1 E7 ratio to be tested after primary calibration/SMN1 E7 ratio normal reference sample after primary calibration].
  • DNA denaturation sequentially add 5 ⁇ l of the above sample DNA to different PCR reaction tubes, and the total amount of all samples loaded is 60ng.
  • the DNA denaturation conditions are as follows, the instrument used in the reaction is Bio-Rad S1000 Thermal Cycler:
  • the hybridization reaction conditions are as follows, the instrument used in the reaction is Bio-Rad S1000 Thermal Cycler:
  • connection reaction system is as follows:
  • connection reaction conditions are as follows, the instrument used in the reaction is Bio-Rad S1000 Thermal Cycler:
  • the PCR reaction conditions are as follows:
  • Example 2 Experimental test of calibration accuracy and intra-batch precision of the SMN gene nucleic acid flight mass spectrometry quantification scheme
  • the Philadelphia saliva sample genome extraction kit was used to extract DNA from saliva samples of known normal volunteers, and after extraction, the nucleic acid concentration was determined by Thermo Fisher Qubit 4.0.
  • Sample detection Using 10ng, 20ng, 40ng, and 80ng DNA as the template respectively, 15 repeated reaction wells are made for each of the 4 loading volumes (15 repeated reactions are completed in 3 batches, and each batch is made 5 repeats); The competitor mixture is sequentially added to the reaction wells as a competitive template, and the sample volume is 2 ⁇ L.
  • the SAP reaction system configuration is as follows:
  • the SAP reaction program settings are as follows:
  • the configuration of the extension reaction system is as follows:
  • the extension reaction program settings are as follows:
  • the 20ng, 40ng, and 80ng loading reactions can all obtain more accurate copy numbers.
  • the calculated copy number for the 10ng loading reaction is A small number of critical values (such as the calculation result of 1.4-1.6 copies) appear
  • this experimental program has good reproducibility and depends on the design of the internal reference DNA sequence. This program has a lot of flexibility in the requirements for the amount of DNA loaded. We have a stable and good DNA extraction program for samples, and the output quality is reliable. At this time, only rough quantification by nanodrop is required.
  • Table 3 The coefficient of variation of intra-assay accuracy of SMN nucleic acid flight mass spectrometry
  • Example 3 Competitor stability test (long-term storage test, repeated freeze-thaw test)
  • the detection scheme of the present invention relies on a competitor, we have tested the stability of the competitor, including long-term storage tests and repeated freeze-thaw tests.
  • the samples used in this test are 2 reference samples, and the loading amount is both 40ng.
  • Sample detection use 40ng gDNA as template; add a mixture of competitors (containing carrier DNA/without carrier DNA) stored under different conditions into the reaction wells as a competitive template in sequence, with a sample volume of 2 ⁇ L.
  • the SAP reaction system configuration is as follows:
  • the SAP reaction program settings are as follows:
  • the configuration of the extension reaction system is as follows:
  • the extension reaction program settings are as follows:
  • the competitor is a synthetic single-stranded primer, which is introduced in this scheme and used as a competitive template for the PCR reaction.
  • the stability of single-stranded DNA is relatively poorer than that of double-stranded DNA. This experiment tested whether the competitor mixture under different storage conditions would affect the copy number quantification scheme.
  • test results of the same sample under different storage conditions are not different, and all can be accurately quantified.
  • test results of the three batches have no difference, and all can be accurately quantified.
  • the competitor mixture configured in this scheme has good stability, and the accuracy of the copy number quantification scheme can be guaranteed under the condition of long-term storage under suitable conditions.
  • the following table shows the competitor contained in the mixed solution and its concentration.
  • QIAGEN Salmon Sperm DNA (its final concentration in the mixture is 100ng/ ⁇ l) is added.
  • Sample detection use 40ng DNA as template; add a mixture of different freeze-thaw times (containing carrier DNA/without carrier DNA) into the reaction wells as a competitive template in sequence, with a sample volume of 2 ⁇ L.
  • the SAP reaction system configuration is as follows:
  • the SAP reaction program settings are as follows:
  • the configuration of the extension reaction system is as follows:
  • the extension reaction program settings are as follows:
  • the competitor is a synthetic single-stranded primer, which is introduced in this scheme and used as a competitive template for the PCR reaction.
  • single-stranded DNA has relatively poor stability, and repeated freezing and thawing may accelerate DNA degradation. This experiment tested whether different freezing and thawing times will affect the accuracy of copy number quantification on the competitor mixture configured in this experimental scheme.
  • the competitor mixture configured in this experiment can still accurately detect the copy number after 20 repeated freezing and thawing. And there is no difference in copy number quantification results between competitor mixtures with different freeze-thaw times.
  • Sample detection add the sample DNA to be tested or the reference sample DNA to the reaction wells in sequence, with a sample volume of 2 ⁇ L; add the competitor mixture to the reaction wells in sequence as a competitive template, with a sample volume of 2 ⁇ L.
  • the SAP reaction system configuration is as follows:
  • the SAP reaction program settings are as follows:
  • the extension reaction program settings are as follows:
  • step D Process the data obtained in step D to obtain the ratio of the target gene and the competitor in the sample to be tested, including SMN1 E7/competitor, SMN2 E7/competitor, SMN1 E8/competitor, SMN2 E8/ Competitor, HBA1/competitor, HBA2/competitor, HBQ/competitor and internal reference DNA sequence correction ratio QC/competitor.
  • the ratio of SMN1 E7/competitor, SMN2 E7/competitor, SMN1 E8/competitor, SMN2 E8/competitor, HBA1/competitor, HBA2/competitor, HBQ/competitor ratio is adjusted to the ratio QC/competitor to obtain The ratio of each target gene after initial calibration, namely [the ratio of target gene and competitor/correction ratio].
  • step b Calibration between samples: same as step a, obtain the ratio of each target gene in the normal reference sample after initial calibration, and compare the ratio of each target gene in the sample to be tested obtained in step a with the corresponding target gene in the normal reference sample The ratios after the initial calibration are compared to obtain the final calibration ratios of each target gene, such as [SMN1 E7 ratio to be tested after primary calibration/SMN1 E7 ratio normal reference sample after primary calibration].
  • step D Analyze the data in step D to obtain the copy number of the target gene of each sample as shown in Table 4:

Abstract

本发明提供一种基因拷贝数定量检测方法。本发明通过基质辅助激光解吸电离飞行时间质谱技术平台结合单碱基延伸反应和竞争PCR技术检测，后续通过多步校正完成对基因拷贝数的准确定量。本发明还提供用于执行上述方法的试剂盒。

Description

基因拷贝数定量分析 技术领域
本发明涉及基因检测领域，更具体涉及基因拷贝数的定量分析。
背景技术
基因拷贝数异常(copy number variations，CNV)是广泛存在于人类基因组的一种结构变异。基因拷贝数异常最早在病人的基因组中发现，如遗传性疾病患者、肿瘤患者；但后续的研究发现基因拷贝数异常在正常的个体中也广泛存在。虽然拷贝数异常发生的频率相对较低，但拷贝数异常的片段大小从1kb到数个Mb不等，其累及的基因序列长度相对较长，与人类的健康和疾病的有着密切关联。已有研究证明诸多基因拷贝数异常与疾病的发病、疾病表型存在着密切联系。基因拷贝数异常的检测对于疾病的诊断治疗，尤其是某些遗传性疾病(例如脊髓型肌萎缩症及α-地中海贫血)的诊断治疗及其重要。
脊髓性肌萎缩症是婴幼儿致死的主要遗传疾病之一，是一种常染色体隐性遗传的神经退行性疾病，表现为脊髓运动神经元变性，骨骼肌萎缩和全身无力。脊髓性肌萎缩症在中的发病率为1/6000-1/10000，全球不同人群携带者频率为1/40-1/50。
SMN基因(survival motor neuron gene)的检测是脊髓性肌萎缩症临床诊断的关键手段。95％的脊髓性肌萎缩患者是由于位于5号染色体长臂的SMN1基因纯合缺失或杂合缺失伴点突变，无法生成功能性的SMN蛋白，致脊髓运动神经元变性，称为5qSMA。这其中，90～95％的患者为SMN1基因纯合缺失，另有5％～10％左右的患者为SMN1杂合缺失伴点突变型即复合杂合型。此外在SMN1基因所在区域附近有一与之高度同源的基因SMN2(相似度高达99％，极难分辨)，在外显子区域仅存在2个位点的差异(7号外显子840C＞T，8号外显子1239G＞A)，在内含子区域有十数个位点的差异。由于SMN1与SMN2外显子7上的840C＞T的位点差异使得SMN2在转录生成mRNA时发生外显子跳跃，无法正确剪接，仅有10％左右的SMN2的转录产物为全长能翻译成功能性的SMN蛋白。有相关研究表明SMN2拷贝数与脊髓性肌萎缩症患者的表型相关，且当下已上市的治疗脊髓性肌萎缩症的nusinersen 是基于修正SMN2 mRNA的正确剪接，从而生成更多的功能性SMN蛋白起到治疗作用。因此，SMN基因主要检测内容包括：SMN1基因缺失检测、SMN2基因拷贝数检测、SMN1基因致病性点突变的检测。
α-地中海贫血是由于α珠蛋白基因(HBA基因)发生突变导致α肽链合成减少或无法合成，导致血红蛋白的组成成份改变，继而引发慢性溶血和贫血。HBA基因包括两个同源基因HBA1及HBA2，正常人中各2拷贝。HBA1基因和HBA2基因HBA基因缺陷包括HBA1和HBA2的基因缺失或致病性点突变。约80-90％的HBA基因缺陷人群是由于HBA1或HBA2的部分缺失或全缺失导致，10％-20％的HBA基因缺陷人群是由于HBA1或HBA2基因的致病性点突变导致。HBA基因缺陷人群是否发病及疾病的严重程度与与其受累HBA基因的拷贝数相关。此外，由于在这两个基因所在区域存在其他与之同源的相似序列，这对HBA1及HBA2的拷贝数定量造成了一定困难。
鉴于基因拷贝数检测对于疾病诊断及研究的重要意义，如何准确的定量基因拷贝数是首要解决的问题。目前常用于基因拷贝数检测的方法主要包括：多重连接探针检测(multiplex ligation-dependent probe amplification，MLPA)，实时荧光定量PCR(qPCR)等。
多重连接探针扩增技术通过设计多组与不同目的位点(涉及目的基因的外显子或内含子的多个位点，及目的基因相邻序列、及诸多内参基因)结合的探针，每组探针包含左探针(left probe oligo，LPO)及右探针(right probe oligo，RPO)，在探针与目的基因结合后，通过连接酶的作用连接同组的左右探针。每组探针的序列除与目的基因结合的序列(hybridization sequence)外包含相同的引物序列(forward primer sequence&reverse primer sequence)及用于区分不同产物的标签序列(stuffer sequence)(标签序列包含的碱基数目有差异，使得不同组探针最终对应的连接产物的长度不一)。在连接反应完成后，所有的连接产物可以通过同一对PCR引物进行扩增，其后通过毛细管电泳区分不同长度的连接产物，分辨出不同目的位点的产物信息。其后通过两步校正结果分析基因拷贝数：①样本内校正：将各目的基因产物与内参基因产物比较获得其各自的比值(目的基因/内参基因)；②样本间校正：将待测样本各目的基因/内参基因的比值与正常对照样本(各待测基因拷贝数为2)的目的基因/内参基因的比值获得校正后的比值，该比值用 于最后评估各基因的绝对拷贝数。
多重连接探针扩增技术检测流程繁琐，耗时偏长，检测成本较高。另外，由于技术本身的局限，该技术的探针设计比较困难，结果分析较为复杂。在对SMN1致病性点突变的多重检测上受限，设计特异性的多重点突变检测方案较为困难。此外，在样本DNA中目的基因与探针结合区域序列若存在突变位点或SNPs，则可能致连接反应失败，后续无扩增产物，致假阴性结果的产生。
实时荧光定量PCR技术通过在目的基因序列上设计PCR扩增引物及特异性的探针；同时在无拷贝数变异的基因序列上涉及PCR扩增引物及特异性的探针作为内参。在多重的实时荧光定量PCR反应中，选用不同荧光信号分别标记目的基因和内参基因。通过多通道实时荧光定量PCR仪器，实时采集用作标记的荧光信号。在实时荧光定量PCR检测待测样本基因拷贝数时，需同时检测对照样品作为参照(对照样品可为目的基因的人工合成序列与内参基因的人工合成序列按一定比例混合)。计算待测样本目的基因与内参基因Ct值的差值ΔCt_s，计算内参样本目的基因与内参基因Ct值的差值ΔCt_r，其后计算ΔΔCt＝ΔCt_s-ΔCt_r，根据ΔΔCt判读基因拷贝数。
该检测方案的单管反应通量低，多重检测不同基因拷贝数变异较为困难。在存在同源基因序列的检测中，需通过加入特异性阻断探针(如PNA或LNA探针)辅助，阻断同源基因之一，收集未被阻断的基因扩增信号。故不能在单管反应中完成对同源基因的检测。该方案的灵敏性、特异性受限于不同基因检测的设计，不同基因检测设计的灵敏性、特异性不同。当灵敏度欠佳时，虽可准确检测纯合缺失的患者(拷贝数为0)，很难精准的对拷贝数进行定量(1/2/3/4/4以上拷贝)。
因此，本领域需要能够更准确、迅速检测基因拷贝数的方法。
发明内容
本发明通过基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry)技术平台结合单碱基延伸反应、real-competitive PCR技术检测，后续通过多步校正完成对基因拷贝数的准确定量。
该方法包括实验室方法和数据分析方法，可以准确检测人类基因 组中的基因拷贝数变异(包括纯合子/杂合子缺失及重复)。
此外，对于人类基因组中存在的高度同源相似的序列，本技术也能通过这些序列的差异碱基设计检测方案，通过单碱基延伸反应可以区分差异碱基，同时完成同源序列的定量。
应用本发明可在单管反应中完成SMN1基因缺失检测、SMN2基因拷贝数检测、HBA1及HBA2的拷贝数定量。同时，核酸飞行质谱平台现已实现自动化，故本发明便于在临床推广应用，且能准确、全面的分析基因拷贝数变化，应用价值高。
具体地，本发明包括以下实施方案：
一方面，本发明涉及检测目的基因拷贝数的方法，包括：
a)设计所述目的基因的扩增和延伸引物、所述目的基因的竞争物、不存在拷贝数变异的内参基因的扩增和延伸引物，和所述内参基因的竞争物，其中所述目的基因的竞争物与所述目的基因存在至少1个核苷酸的差异并且与所述目的基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性，作为与所述目的基因的竞争性扩增模板在PCR扩增反应中同时扩增，所述目的基因的延伸引物在延伸反应中能够分别延伸所述目的基因和目的基因的竞争物的扩增产物；所述内参基因的竞争物与所述内参基因存在至少1个核苷酸序列的差异并且与所述内参基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性，作为所述内参基因的竞争性扩增模板在PCR扩增反应中同时扩增，所述内参基因的延伸引物能够在延伸反应中分别延伸所述内参基因和内参基因的竞争物的扩增产物；
b)在获自怀疑具有目的基因拷贝数变异的受试者的待测DNA样本和获自正常人的正常对照DNA样本中加入步骤a)所述的扩增引物，进行PCR扩增，得到目的基因、内参基因及其各自竞争物的PCR扩增产物；
c)在所述PCR扩增产物中加入步骤a)所述的延伸引物，进行延伸反应，得到目的基因、内参基因及其各自竞争物的延伸产物；
d)对所述延伸产物进行基质辅助激光解吸电离飞行时间质谱，并且将得到的各个延伸产物的数据进行以下分析：
i)样本内校正：计算待测样本中目的基因的校正用比值，即，内 参基因/内参基因竞争物的比值，并且获得目的基因/目的基因竞争物的比值；用目的基因/目的基因竞争物的比值除以校正用比值，得到目的基因的初校后比值，即：目的基因与目的基因竞争物的比值/内参基因与内参基因竞争物的比值；
ii)样本间校正：按照步骤i)的方法，计算正常对照样本中目的基因的初校后比值；将步骤i)中获取的待测样本中的目的基因初校后比值与正常对照样本中相应的目的基因初校后比值相比，获得目的基因的终校后比值，即目的基因初校后比值 待测样本/目的基因初校后比值 正常参考样本；
iii)用待测样本目的基因终校后比值乘以2，得到待测样本中目的基因的拷贝数。
另一方面，本发明涉及检测目的基因拷贝数的试剂盒，其中含有所述目的基因的扩增和延伸引物、所述目的基因的竞争物、不存在拷贝数变异的内参基因的扩增和延伸引物，和所述内参基因的竞争物，其中所述目的基因的竞争物与所述目的基因存在至少1个核苷酸的差异并且与所述目的基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性，所述内参基因的竞争物与所述内参基因存在至少1个核苷酸的差异并且与所述内参基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性。
在一个实施方案中，DNA样本选自体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、肺灌洗液、脑脊液、粪便和组织样本。
在一个实施方案中，所述目的基因的竞争物与所述目的基因存在1、2、3、4、5、6、7、8、9或10个核苷酸的差异，并且所述内参基因的竞争物与所述内参基因存在1、2、3、4、5、6、7、8、9或10个核苷酸的差异。
在一个实施方案中，所述目的基因是SMN1、SMN2、NAIP、HBA1、HBA2和/或HBQ基因。
在一个实施方案中，所述目的基因的扩增和延伸引物选自i)SMN17号外显子和SMN2 7号外显子的扩增和延伸引物，其分别如SEQ ID NO：1-2和SEQ ID NO：6所示；ii)SMN1 8号外显子和SMN2 8号外显子 的扩增和延伸引物，其分别如SEQ ID NO：3-4和SEQ ID NO：5所示；iii)NAIP基因5号外显子的扩增和延伸引物，其分别如SEQ ID NO：13-14和SEQ ID NO：15所示；iv)HBA1基因的扩增和延伸引物，其分别如SEQ ID NO：17-18和SEQ ID NO：24所示；v)HBA2基因的扩增和延伸引物，其分别如SEQ ID NO：19-20和SEQ ID NO：26所示；和vi)HBQ基因的扩增和延伸引物，其分别如SEQ ID NO：21-22和SEQ ID NO：28所示。
在一个实施方案中，所述目的基因的竞争物选自i)SMN17号外显子和SMN2 7号外显子的竞争物，其如SEQ ID NO：7所示；ii)SMN1 8号外显子和SMN2 8号外显子的竞争物，其如SEQ ID NO：8所示；iii)NAIP基因5号外显子的竞争物，其如SEQ ID NO：16所示，iv)HBA1基因的竞争物，其如SEQ ID NO：23所示，v)HBA2基因的竞争物，其如SEQ ID NO：25所示，和vi)HBQ基因的竞争物，其如SEQ ID NO：27所示。
在一个实施方案中，所述内参基因是NFATC3基因，所述内参基因的扩增和延伸引物分别如SEQ ID NO：9-10和11所示，所述内参基因的竞争物如SEQ ID NO：12所示。
另一方面，本发明涉及引物，其如选自SEQ ID NO：1-6、SEQ ID NO：9-11、SEQ ID NO：13-15、SEQ ID NO：17-22、SEQ ID NO：24、SEQ ID NO：26和SEQ ID NO：28的序列所示。
另一方面，本发明涉及多核苷酸，其如选自SEQ ID NO：7、8、12、16、23、25和27的序列所示。
本发明可准确检测基因拷贝数变异，且同样可以应用于高度同源基因的精确拷贝数检测。
本发明可以在同一平台进行基因拷贝数的检测，提供疾病和健康相关基因的更全面分析。
不同于其他拷贝数变异检测方案，需有精准的DNA定量技术定量后，将所有待测样本与正常参考样本上样量统一。本发明可在DNA提取后，可通过nanodrop直接定量，甚至无需DNA定量步骤，理想待测样本上样量范围可为10-80ng，正常参考样本可选择在此区间任何量，比如10ng、20ng或者40ng用于后续校正即可，进一步精简操作，并降低检测成本。
本发明所用核酸飞行质谱平台已自动化，流程简单，耗时相对较少(8小时左右)，便于在临床推广应用。可通过本发明设计诸多与疾病相关的拷贝数变异检测方案，并在临床上实际推广应用。
附图说明
图1是SMN1、SMN2、NAIP基因拷贝数定量检测方案及预期结果示意图：以SMN1基因7号外显子上的设计为例，在SMN1基因7号外显子和SMN2基因8号外显子的差异碱基上下游设计PCR扩增引物，对该段序列进行扩增。此外，在PCR扩增时除gDNA模板外引入竞争物作为竞争性模板，竞争物的序列除差异碱基外，与gDNA扩增子的序列相同。基于上述差异碱基，进行了延伸引物的设计，不同的碱基之间分子量有差异，后续通过单碱基延伸反应和核酸飞行质谱检测平台识别区分SMN1、SMN2、竞争物的差异碱基。如结果示意图所示，可以区分不同SMN1基因拷贝数、不同SMN2基因拷贝数的人群，区分正常人、携带者及患者。
图2显示SMN1、SMN2、NAIP基因拷贝数定量检测方案在临床样本(正常、携带者、患者)中SMN1基因7号外显子检测结果及SMN2基因8号外显子中的检测结果。达成预期的检测效能，对正常、携带者、患者三类人群能准确区分诊断。
图3显示SMN1基因、SMN2基因、NAIP基因拷贝数定量方案拷贝数校正测试结果：目前其他拷贝数定量的检测方案大多要求较精准一致的模板上样量，而本方案的设计方案和校正方案对模板上样量的要求有较大的弹性空间。分别以10ng、20ng、40ng、80ng上样量做了15个重复。图3A，以随机4个10ng上样反应作为正常参考样本校正时的各反应拷贝数计算结果；图3B，以随机4个20ng上样反应作为正常参考样本校正时的各反应拷贝数计算结果；图3C，以随机4个40ng上样反应作为正常参考样本校正时的各反应拷贝数计算结果；图3D，以随机4个80ng上样反应作为正常参考样本校正时的各反应拷贝数计算结果。结果显示在以20ng上样反应作为正常参考样本校正时，10ng、20ng、40ng上样反应均能获得较为准确的拷贝数结果。
图4A-4D显示竞争物混合液储存稳定性测试结果：应用不同储存条件下竞争混合液进行了拷贝数定量检测方案的检测。图4A，应用添加了载体DNA，-20℃储存条件下竞争混合液的拷贝数定量结果；图4B，应用添加了载体DNA，4℃储存条件下竞争混合液的拷贝数定量结果；图4C，应用未添加载体DNA，-20℃储存竞争混合液的拷贝数 定量结果；图4D，应用未添加了载体DNA，4℃储存竞争混合液的拷贝数定量结果。结果显示上述4种储存条件下，竞争物混合液均具有良好的稳定性，应用于拷贝数定量检测方案时，能稳定准确地对拷贝数进行定量。
图5A-5C显示竞争物反复冻融测试结果：对竞争物混合液进行多次冻融测试模拟在实际使用中的多次冻融，测试其在多次反复冻融后的稳定性。对竞争物混合液进行20次反复冻融，并应用其不同冻融次数的竞争物混合液做拷贝数定量检测方案的测试。图5A、5B，应用不同冻融次数的添加了载体DNA的竞争物混合液做拷贝数定量检测结果；图5C，应用不同冻融次数的未添加载体DNA的竞争物混合液做拷贝数定量检测结果。结果显示添加和未添加载体DNA的竞争物混合液在反复冻融中均稳定性良好，均能准确进行拷贝数定量检测。
图6显示HBA基因拷贝数检测方案设计示意图：针对HBA基因常见三种缺失型，在HBA1基因和HBA2基因附近选取了三个高度保守的区域作为靶点进行了检测方案的设计。如图所示，αα 4.2缺失型仅缺失HBA2基因上游靶点 HBA2，αα 3.7缺失型仅缺失HBA1基因上游靶点H BA1，-- SEA缺失型同时缺失HBA2基因上游靶点 HBA2、HBA1基因上游靶点 HBA1，及HBQ基因上游靶点 HBAQ。
具体实施方式
现以SMN基因检测为例，对本发明的技术方案进行举例说明：
设计方案：
A.95％左右的脊髓性肌萎缩症患者的SMN1基因存在外显子7和/或外显子8的缺失。故本发明针对SMN基因外显子7及外显子8分别设计了PCR扩增引物，同时扩增SMN1及SMN2外显子7/8区域(扩增区域包含SMN1与SMN2外显子7上的差异位点，即7号外显子C840T，8号外显子G1239A)。针对SMN1和SMN2的两个差异位点所在分别设计了延伸引物，故在后续的单碱基延伸反应中可以区分SMN1和SMN2，通过和核酸飞行质谱平台可获得SMN1/SMN2，完成SMN1和SMN2的相对定量。
PCR引物命名及序列如下：
SMN_c.C840T_F：AACTTCCTTTATTTTCCTTAC(SEQ ID NO：1)
SMN_c.C840T_R：TAATGCTGGCAGACTTACTCC(SEQ ID NO：2)
SMN_c.G1239A_F：GTGAAATATTTTACTGGACTC(SEQ ID NO：3)
SMN_c.G1239A_R：TTTTCTCAACTGCCTCACCAC(SEQ ID NO：4)
延伸引物命名及序列如下：
SMN_c.G1239A_U：CCTCCCACCCCCACC(SEQ ID NO：5)
SMN_c.C840T_U：TTTATTTTCCTTACAGGGTTT(SEQ ID NO：6)
B.设计至少2个竞争物序列，分别对应步骤A所设计的两对PCR引物所扩增的目的序列，在以下位点(C840T＞G，G1239A＞C)分别引入序列变异(下划线标注碱基为引入的变异碱基)。
SMN_c.C840T_C：(95bp)
Figure PCTCN2020086108-appb-000001
SMN_c.G1239A_C：(95bp)
Figure PCTCN2020086108-appb-000002
C.为更加准确对SMN1和SMN2拷贝数的绝对定量，选取人类基因组上不存在拷贝数变异的基因序列作为内参。该段序列为位于人类16号染色体NFATC3基因外显子2上的一段序列，我们通过DECIPHER v9.30 database(该数据库涉包含30405人的基因组数据)选取的一段在其数据库人群中未见拷贝数变异的序列，并后续在UCSC的CNV database中确认该段序列确无拷贝数变异的记录。作为内参用于进一步的校正。选取的内参序列PCR扩增引物及延伸引物序列如下：
QC_g.G3C_F：ATATAGCCCATTAGGTGGTCC(SEQ ID NO：9)
QC_g.G3C_R：CTGTAGGTCATCTTCATGTGC(SEQ ID NO：10)
QC_g.G3C_U：TTGAATACTTGGGCACT(SEQ ID NO：11)
同时，如步骤B对该段扩增子待测位点引入不同于参考序列的碱基设计竞争物，其命名及序列如下：
QC_g.G3C_C：(109bp)
Figure PCTCN2020086108-appb-000003
Figure PCTCN2020086108-appb-000004
同步骤C中描述，在后续延伸反应中，基因组DNA模板和竞争物模板对应的延伸产物可通过核酸飞行质谱平台区分，并获得QC/竞争物用于后续校正，我们称之为内参校正比值。
D.有研究表明，脊髓性肌萎缩症患者的表型与NAIP基因拷贝数存在相关性，故如步骤D中QC测定的设计，我们对NAIP基因外显子5区域应用同样的方法进行了设计。其PCR扩增引物、延伸引物及竞争物序列分别如下：
NAIP-E5_c.G5C_F：GGAACCATTTGGCATGTTCCT(SEQ ID NO：13)
NAIP-E5_c.G5C_R：ACAGTGTTTTTCCTGTGGTGG(SEQ ID NO：14)
NAIP-E5_c.G5C_U：TGGGAAGAAGGAGATGAT(SEQ ID NO：15)
NAIP-E5_c.G5C_C：(81bp)
Figure PCTCN2020086108-appb-000005
E.样本内校正：(1).我们通过步骤A-D中相应竞争物的设计，可在各设计中获得目的基因和竞争物的比值，包括SMN1 E7/竞争物、SMN2 E7/竞争物、SMN1 E8/竞争物、SMN2 E8/竞争物、NAIP E5/竞争物及内参DNA序列的校正用比值QC/竞争物。在各反应中加入的竞争物为固定量，故上述目的基因比值和校正用比值可反映目的基因和内参基因的绝对量，且通过该比值使得我们在后续校正时减小了由于样本本身差异对PCR扩增反应的影响，定量更加准确。(2).其后通过将样本中获得的各目的基因比值与内参DNA序列(拷贝数稳定且正常的序列)的校正用比值相比，获得各目的基因在样本中的初校后比值。有赖于竞争物的设计，此时所获得的初校后比值避免了因目的序列与内参DNA序列PCR反应扩增效率可能不同造成的影响。(而在MLPA的设计中不存在类似竞争物的设计，使得其在样本内校正时只能获得目的基因/内参基因，若需减少样本差异或不同扩增效率差异造成的影响则需对数据进行更繁琐的校正。)注：此处样本包括待测的未知样本和3～4个用于进一步校正的已知拷贝数正常的参考样本。
F.样本间校正：在步骤A-F中我们获取了待测样本和一个或者多个正常参考样本的各目的基因初校后比值。将待测样本中的初校后比值与正常参考样本中的初校后比值相比获得各目的基因的终校后比值，如[SMN1 E7初校后比值 待测样本/SMN1 E7初校后比值 正常参考样本]。同时，已知正常参考样本中目的基因拷贝数均为2，故各待测样本的目的基因拷贝数即为各目的基因终校后比值*2，如SMN1 E7拷贝数＝[SMN1 E7初校后比值 待测样本/SMN1 E7初校后比值 正常参考样本]*2。此外，有赖于竞争物联合内参DNA序列检测的设计，DNA上样量差异造成的影响在步骤F中已被大幅减少。故仅需保证待测样本上样量在一定区间内，同时选取在该区间内合适上样量的正常参考样本数据进行校正即可获得这些上样量不一的未知样本目的基因拷贝数。在样本类型及抽提DNA的方法确定且稳定的情况下，对待测样本进行粗糙的nanodrop定量甚至不需要预先定量，可通过设定多个不同上样量的正常样本用作校正即可获取待测样本目的基因拷贝数。(而在MLPA中，在同批实验中必须保证各待测样本与正常参考样本上样量均一致方可实验。)
本方案针对SMN1基因和SMN2基因的7号外显子和8号外显子的差异碱基进行了检测方案的设计(图1)，本方案能准确区分不同SMN1拷贝数、不同SMN2拷贝数的人群(图2)。
本拷贝数定量检测方案可应用于更多的机制明确的缺失型缺陷的单基因疾病。α-地中海贫血为HBA基因缺陷所致，90％的HBA基因缺陷为缺失型，故本拷贝数定量检测方案同样可应用于HBA基因缺失型的检测。故我们在完善的SMN基因检测方案的基础上，增添了HBA基因的拷贝数检测方案。
SMN基因拷贝数定量检测部分如上执行方案所述，增添的HBA基因拷贝数检测方案如下：
A.50％左右的HBA基因缺陷携带者为SEA缺失型αα/-- SEA，15％左右的HBA基因缺陷携带者为3.7缺失型(αα/αα 3.7)，15％左右的HBA基因缺陷携带者为4.2缺失型(αα/αα 4.2)，此外还有约10％左右的患者为SEA缺失型、3.7缺失型、4.2缺失型缺陷基因的复合型(如：-- SEA/αα 4.2，-- SEA/αα 3.7等)。不同于SMN基因检测方案设计思路，本方案在HBA2基因上游、HBA1基因上游、HBQ基因上游的特异性序列上进行了检测方案的设计。
B.在保证前述SMN检测方案的特异性的同时，本方案在HBA2基因上游、HBA1基因上游、HBQ基因上游的特异性序列上进行了检测方案的设计，故对这三个区域的PCR扩增引物设计如下。
HBA1_F：ACGTTGGATGTCAGCACCCTTCAGCCTGCTC(SEQ ID NO：17)
HBA1_R：ACGTTGGATGTTCTCTGCCCAAGGCAGCTTA(SEQ ID NO：18)
HBA2_F：ACGTTGGATGGAGACACTTCACTGAGAATAGG(SEQ ID NO：19)
HBA2_R：ACGTTGGATGATCTACAACTACTGCCACAGG(SEQ ID NO：20)
HBQ_F：ACGTTGGATGTGCCATAGGTGTTTACCAAGG(SEQ ID NO：21)
HBQ_R：ACGTTGGATGAGCTGGTAGCCATAAAGCCCTG(SEQ ID NO：22)
C.在上述PCR引物所扩增序列中选取合适的碱基突变(下划线标注)，进行竞争物及延伸引物的设计，设计如下：
HBA1_C(115bp)：
Figure PCTCN2020086108-appb-000006
HBA1_U：AGGCAGCTTACCCTGG(SEQ ID NO：24)
Figure PCTCN2020086108-appb-000007
HBA2_U：tCTCTCTTTTTGGACAAAAATAC(SEQ ID NO：26)
Figure PCTCN2020086108-appb-000008
HBQ_U：agtAATATCTTTTATTCCCTGAGC(SEQ ID NO：28)
D.样本内校正：(1).我们通过竞争物的设计，可在各设计中获得目的基因和竞争物的比值，包括SMN1 E7/竞争物、SMN2 E7/竞争物、 SMN1 E8/竞争物、SMN2 E8/竞争物、HBA1/竞争物、HBA2/竞争物、HBQ/竞争物及内参DNA序列的校正用比值QC/竞争物。在各反应中加入的竞争物为固定量，故上述目的基因比值和校正用比值可反映目的基因和内参基因的绝对量，且通过该比值使得我们在后续校正时减小了由于样本本身差异对PCR扩增反应的影响，定量更加准确。(2).其后通过将样本中获得的各目的基因比值与内参DNA序列(拷贝数稳定且正常的序列)的校正用比值相比，获得各目的基因在样本中的初校后比值。有赖于竞争物的设计，此时所获得的初校后比值避免了因目的序列与内参DNA序列PCR反应扩增效率可能不同造成的影响。(而在MLPA的设计中不存在类似竞争物的设计，使得其在样本内校正时只能获得目的基因/内参基因，若需减少样本差异或不同扩增效率差异造成的影响则需对数据进行更繁琐的校正。)注：此处样本包括待测的未知样本和3～4个用于进一步校正的已知拷贝数正常的参考样本。
E.样本间校正：在步骤A-E中我们获取了对未知待测样本和一个或者多个正常参考样本进行分析获得的各目的基因初校后比值与样本中各目的基因拷贝数成正比。故已知正常样本中各目的基因拷贝数为2，通过将未知样本中的初校后比值与正常参考样本中的初校后比值相比获得各目的基因的终校后比值，如[SMN1 E7初校后比值待测样本/SMN1 E7初校后比值正常参考样本]。同时，已知正常参考样本中目的基因拷贝数均为2，故各待测样本的目的基因拷贝数即为各目的基因终校后比值*2，如SMN1 E7拷贝数＝[SMN1 E7初校后比值待测样本/SMN1 E7初校后比值正常参考样本]*2。，即可获得未知样本中各目的基因拷贝数。此外，有赖于竞争物联合内参DNA序列检测的设计，DNA上样量差异造成的影响在步骤E中已被大幅减少。故仅需保证未知待测样本上样量在一定区间内，同时选取在该区间内合适上样量的正常参考样本数据进行校正即可获得这些上样量不一的未知待测样本目的基因拷贝数。在样本类型及抽提DNA的方法确定且稳定的情况下，对未知待测样本进行粗糙的nanodrop定量甚至不需要预先定量，可通过设定多个不同上样量的正常样本用作校正即可获取未知待测样本目的基因拷贝数。(而在MLPA中，在同批实验中必须保证各未知待测样本与正常参考样本上样量均一致方可实验。)
上述实验方案设计见图6以及下表：
Figure PCTCN2020086108-appb-000009
实施例1：应用本发明方案与MLPA方案同时进行拷贝数定量测试并比较
核酸提取
收取确诊为脊髓性肌萎缩症患者2例、确诊为脊髓性肌萎缩症携带者5例、确诊为正常5例，及未知结果66例孕妇外周血样本(DNA已提取)。使用Qiagen Blood Mini Kit提取EDTA抗凝管所采集的全血样本。提取后采用Thermo Fish Picogreen DNA定量assay测定核酸浓度，并调整至20ng/ul。参考样本采用相同定量方案定量后调整浓度至20ng/ul。
核酸飞行质谱方案检测
A.PCR反应(试剂盒：Agena PCR reagent set)：
(1)配置拷贝数检测PCR引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000010
(2)配置竞争物混合液，下表为该混合液中包含的竞争物及其浓度
Figure PCTCN2020086108-appb-000011
(3)多重PCR反应体系配置，总反应体积10μL，如下表(试剂盒：Agena PCR reagent set)
Figure PCTCN2020086108-appb-000012
样本检测：将待测样本DNA或参考样本DNA依序加入反应孔位，加样体积2μL；将竞争物混合液依序加入反应孔位作为竞争性模板，加样体积为2μL。
PCR反应程序设置如下表：
Figure PCTCN2020086108-appb-000013
PCR反应完成后依序转移5μL PCR产物至新的反应板中，用于后续的虾碱性磷酸酶(SAP)反应。
B.SAP反应(试剂盒：Agena iPLEX pro reagent set)：
SAP反应体系配置如下表：
Figure PCTCN2020086108-appb-000014
向步骤A中最后所得的5μL PCR产物依序加入2μL SAP反应液。
SAP反应程序设置如下表：
Figure PCTCN2020086108-appb-000015
C.延伸反应(试剂盒：Agena iPLEX pro reagent set)：
(1)配置拷贝数检测延伸引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000016
(2)延伸反应体系配置如下表：
Figure PCTCN2020086108-appb-000017
Figure PCTCN2020086108-appb-000018
延伸反应程序设置如下表：
Figure PCTCN2020086108-appb-000019
D.核酸飞行质谱平台点样分析获取数据并分析，具体如下。
a)样本内校正：处理步骤D中获得的数据，获得待测样本中的目的基因和竞争物的比值，包括SMN1 E7/竞争物、SMN2 E7/竞争物、SMN1 E8/竞争物、SMN2 E8/竞争物、NAIP E5/竞争物及内参DNA序列的校正用比值QC/竞争物。将SMN1 E7/竞争物、SMN2 E7/竞争物、SMN1 E8/竞争物、SMN2 E8/竞争物、NAIP E5/竞争物比上校正用比值QC/竞争物，获得各目的基因的初校后比值，即[目的基因和竞争物比值/校正用比值]。
b)样本间校正：同步骤a，获取正常参考样本中的各目的基因初校后比值，将步骤a中获取的待测样本中各目的基因初校后比值与正常参考样本中相应的目的基因初校后比值相比，获得各目的基因终校后比值，如[SMN1 E7初校后比值 待测样本/SMN1 E7初校后比值 正常参考样本]。
c)已知正常参考样本中目的基因拷贝数均为2，故各待测样本的目的基因拷贝数即为各目的基因终校后比值*2，如SMN1 E7拷贝数＝[SMN1 E7初校后比值 待测样本/SMN1 E7初校后比值 正常参考样本]*2。
MLPA方案检测
试剂盒：SALSA MLPA P021 SMA probe mix
DNA变性：依序向不同PCR反应管中各加入5μl上述样本DNA，所有样本上样总量均为60ng。
DNA变性条件如下表，反应所使用仪器为Bio-Rad S1000 Thermal Cycler：
Figure PCTCN2020086108-appb-000020
A.杂交反应体系配置如下表：
Figure PCTCN2020086108-appb-000021
杂交反应条件如下表，反应所使用仪器为Bio-Rad S1000 Thermal Cycler：
Figure PCTCN2020086108-appb-000022
B.连接反应体系配置如下表：
Figure PCTCN2020086108-appb-000023
连接反应条件如下表，反应所使用仪器为Bio-Rad S1000 Thermal Cycler：
Figure PCTCN2020086108-appb-000024
C.PCR反应体系配置如下表，反应所使用仪器为Bio-Rad S1000  Thermal Cycler：
Figure PCTCN2020086108-appb-000025
PCR反应条件如下表：
Figure PCTCN2020086108-appb-000026
D.通过ABI 3730xl毛细管电泳分析PCR产物获得数据，并应用MRC-hollad公司提供的coffalyser.Net程序分析数据。
结果分析：
表一、12例已知样本核酸飞行质谱方案检测结果与MLPA方案检测结果比较
Figure PCTCN2020086108-appb-000027
表二、66例孕妇外周血样本核酸飞行质谱方案与MLPA方案结果比较：
Figure PCTCN2020086108-appb-000028
结论：本发明提供的核酸飞行质谱方案检测结果与MLPA方案一致。
实施例2：SMN基因核酸飞行质谱定量方案校正精度及批内精密度实验测试
核酸提取
使用apostle唾液样本基因组提取试剂盒提取已知正常志愿者唾液样本DNA，提取后采用Thermo Fisher Qubit 4.0测定核酸浓度。
核酸飞行质谱方案检测
A.PCR反应(试剂盒：Agena PCR reagent set)：
(1)配置拷贝数检测PCR引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000029
(2)配置竞争物混合液，下表为该混合液中包含的竞争物及其浓度
Figure PCTCN2020086108-appb-000030
(3)多重PCR反应体系配置，总反应体积10μL，如下表：
Figure PCTCN2020086108-appb-000031
样本检测：分别以10ng、20ng、40ng、80ngDNA作为模板，4个上样量均做15个重复反应孔(15次重复反应分3个批次完成，每个批次做5个重复)；将竞争物混合液依序加入反应孔位作为竞争性模板，加样体积为2μL。
PCR反应程序设置如下表：
Figure PCTCN2020086108-appb-000032
PCR反应完成后依序转移5μL PCR产物至新的反应板中，用于后续的SAP反应。
B.SAP反应：
SAP反应体系配置如下表：
Figure PCTCN2020086108-appb-000033
Figure PCTCN2020086108-appb-000034
向步骤A中最后所得的5μL PCR产物依序加入2μL SAP反应液。
SAP反应程序设置如下表：
Figure PCTCN2020086108-appb-000035
C.延伸反应：
配置拷贝数检测延伸引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000036
延伸反应体系配置如下表：
Figure PCTCN2020086108-appb-000037
延伸反应程序设置如下表：
Figure PCTCN2020086108-appb-000038
D.核酸飞行质谱平台点样分析获取数据并分析，在获取各反应的目的基因初校比值后，随机选取4个10ng上样反应作为正常参考样本校正，计算其他所有反应中各目的基因的拷贝数。随后依次以20ng、40ng、80ng上样反应中的随机4个作为正常参考样本校正，计算其他所有反应中各目的基因的拷贝数。4次拷贝数计算结果见图3A-D。
如图3A-D所示：
1.在相同上样量的反应中，各目的基因拷贝数计算结果一致，且批间重复性亦良好；
2.在10ng、20ng、40ng、80ng不同上样量的情况下，各目的基因拷贝数计算结果分布模式一致；
3.如图3B所示，当以20ng上样反应作为正常参考样本校正时，10ng、20ng、40ng上样的反应均能获得较为准确的拷贝数，此时80ng上样的反应所计算的拷贝数有相对多的临界值(如计算结果为2.4-2.6拷贝)出现。
如图3C所示，当以40ng上样反应作为正常参考样本校正时，20ng、40ng、80ng上样的反应均能获得较为准确的拷贝数，此时10ng上样的反应所计算的拷贝数有少量的临界值(如计算结果为1.4-1.6拷贝)出现
4.综上，本实验方案具有良好的重复性，且有赖于内参DNA序列的设计，该方案对DNA上样量的要求有很大弹性空间。我们对于样本有稳定良好的DNA提取方案，产出质量均可靠，此时仅需通过nanodrop粗略定量即可。
表三：SMN核酸飞行质谱定量批内精度变异系数
Figure PCTCN2020086108-appb-000039
实验结论：上述数据验证了竞争物联合内参DNA序列的实验设计及后续的校正方法可大幅减少上样量差异对拷贝数定量带来的影响。且从上述数据来看，在后续实验中选取10ng、20ng、40ng上样的正常样本用作校正在获取准确的结果的同时，减少了对DNA上样量的准确性的要求。此外，在15次重复中，批内变异系数均＜12％，反应稳定且具有良好的重复性。
实施例3：竞争物稳定性测试(长期储存测试、反复冻融测试)
由于本发明检测方案所依托于竞争物，我们对竞争物的稳定性进行了测试，包括长期储存测试和反复冻融测试。
一、长期储存测试：
1.配置2管具有载体DNA(carrier DNA)的竞争物混合物和2管不具有载体DNA的竞争物混合物分别储存于-20℃、4℃，并在各自管子上标记其储存温度。
配置竞争物混合液，下表为该混合液中包含的竞争物及其浓度。 同时为测试加入载体DNA是否能增强其储存的稳定性，加入QIAGEN Salmon Sperm DNA(其在混合液中终浓度为100ng/μl)。
Figure PCTCN2020086108-appb-000040
2.每隔一段时间取出并应用核酸飞行质谱检测方案检测测试其稳定性，分析比较不同时间点各个储存温度下的测试结果。
3.该测试所用样本为2个参考样本，上样量均为40ng。
4.应用前述核酸飞行质谱检测方案检测。
A.PCR反应(试剂盒：Agena PCR reagent set)：
(1)配置拷贝数检测PCR引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000041
(2)多重PCR反应体系配置，总反应体积10μL，如下表：
Figure PCTCN2020086108-appb-000042
Figure PCTCN2020086108-appb-000043
样本检测：以40ng gDNA作为模板；将不同条件下储存的竞争物混合液(含载体DNA/不含载体DNA)依序加入反应孔位作为竞争性模板，加样体积为2μL。
PCR反应程序设置如下表：
Figure PCTCN2020086108-appb-000044
PCR反应完成后依序转移5μL PCR产物至新的反应板中，用于后续的SAP反应。
D.SAP反应(试剂盒：Agena iPLEX pro reagent set)：
SAP反应体系配置如下表：
Figure PCTCN2020086108-appb-000045
向步骤A中最后所得的5μL PCR产物依序加入2μL SAP反应液。
SAP反应程序设置如下表：
Figure PCTCN2020086108-appb-000046
E.延伸反应(试剂盒：Agena iPLEX pro reagent set)：
配置拷贝数检测延伸引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000047
延伸反应体系配置如下表：
Figure PCTCN2020086108-appb-000048
延伸反应程序设置如下表：
Figure PCTCN2020086108-appb-000049
5.核酸飞行质谱平台点样分析获取数据并分析
6.结果分析见图4A-4D。
竞争物为人工合成的单链引物，本方案将其引入用作PCR反应的竞争性模板。理论上单链DNA相较双链DNA其稳定性会相对较差， 本实验测试了不同储存条件下竞争物混合物是否会对本拷贝数定量方案造成影响。
如图4A-D所示：应用不同储存条件(储存温度、是否添加载体DNA)下对同一样本的检测结果无差异，均能准确定量。且在各储存条件下，三个批次(每个批次检测时间相差30天左右)的检测结果无差异，均能准确定量。
故本方案所配置竞争物混合物有良好的稳定性，在合适条件下长期储存的情况下能保证拷贝数定量方案的准确性。
二、反复冻融测试：
1.配置具有载体DNA的竞争物混合物和不具有携带者DNA的竞争物混合物并储存于-80℃。
下表为该混合液中包含的竞争物及其浓度。同时为测试加入载体DNA是否能增强其储存的稳定性，加入QIAGEN Salmon Sperm DNA(其在混合液中终浓度为100ng/μl)。
Figure PCTCN2020086108-appb-000050
2.待完全结冰后，取出置于室温条件下完全解冻。振荡离心，分别吸取10ul至新的管子中，标记为解冻1次，并将其置于-80℃保存。将原始管重新置于-80℃冰冻。
3.待完全结冰后，取出置于室温条件下完全解冻。再将原始管置于-80℃冰冻。
4.待完全结冰后，取出置于室温条件下完全解冻。振荡离心，分别吸取10ul至新的管子中，标记为解冻3次，并将其置于-80℃保存。将原始管重新置于-80℃冰冻。
5.重复3-4数次，获得解冻5、7、9、11、13、15、17、19次的竞争物混合物均置于-80℃保存。
6.将解冻1、3、5、7、9、11、13、15、17、19次的竞争物混合物全部取出，再次解冻获得解冻2、4、6、8、10、12、14、16、 18、20次的竞争物混合物。
7.应用前述核酸飞行质谱检测方案检测。
A.PCR反应(试剂盒：Agena PCR reagent set)：
(1)配置拷贝数检测PCR引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000051
(2)多重PCR反应体系配置，总反应体积10μL，如下表：
Figure PCTCN2020086108-appb-000052
样本检测：以40ngDNA作为模板；将冻融不同次数的竞争物混合液(含载体DNA/不含载体DNA)依序加入反应孔位作为竞争性模板，加样体积为2μL。
PCR反应程序设置如下表：
Figure PCTCN2020086108-appb-000053
Figure PCTCN2020086108-appb-000054
PCR反应完成后依序转移5μL PCR产物至新的反应板中，用于后续的SAP反应。
B.SAP反应(试剂盒：Agena iPLEX pro reagent set)：
SAP反应体系配置如下表：
Figure PCTCN2020086108-appb-000055
向步骤A中最后所得的5μL PCR产物依序加入2μL SAP反应液。
SAP反应程序设置如下表：
Figure PCTCN2020086108-appb-000056
C.延伸反应(试剂盒：Agena iPLEX pro reagent set)：
配置拷贝数检测延伸引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000057
延伸反应体系配置如下表：
Figure PCTCN2020086108-appb-000058
延伸反应程序设置如下表：
Figure PCTCN2020086108-appb-000059
8.结果分析见图5A-5C。
竞争物为人工合成的单链引物，本方案将其引入用作PCR反应的竞争性模板。单链DNA相较双链DNA其稳定性会相对较差，反复冻融可能会加速DNA的降解。本实验测试了不同冻融次数对本实验方案所配置的竞争物混合物是否会最终影响拷贝数定量的准确性。
如图5A-C所示：本实验所配置的竞争物混合物，历经20次反复冻融，仍能准确检测拷贝数。且应用不同冻融次数的竞争物混合物之间的拷贝数定量结果无差异。
实施例4：SMN基因和HBA基因拷贝数的联合检测
核酸提取
采集7名志愿者2ml唾液样本，应用apostle唾液DNA提取试剂盒对唾液样本进行提取。提取后采用Thermo Fish Picogreen DNA定量assay测定核酸浓度，并调整至20ng/ul。参考样本采用相同定量方案定量后调整浓度至20ng/ul。
核酸飞行质谱方案检测
A.PCR反应(试剂盒：Agena PCR reagent set)：
(1)配置拷贝数检测PCR引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000060
(2)配置竞争物混合液，下表为该混合液中包含的竞争物及其浓度
Figure PCTCN2020086108-appb-000061
(4)多重PCR反应体系配置，总反应体积10μL，如下表(试剂盒：Agena PCR reagent set)
Figure PCTCN2020086108-appb-000062
Figure PCTCN2020086108-appb-000063
样本检测：将待测样本DNA或参考样本DNA依序加入反应孔位，加样体积2μL；将竞争物混合液依序加入反应孔位作为竞争性模板，加样体积为2μL。
PCR反应程序设置如下表：
Figure PCTCN2020086108-appb-000064
PCR反应完成后依序转移5μL PCR产物至新的反应板中，用于后续的虾碱性磷酸酶(SAP)反应。
B.SAP反应(试剂盒：Agena iPLEX pro reagent set)：
SAP反应体系配置如下表：
Figure PCTCN2020086108-appb-000065
向步骤A中最后所得的5μL PCR产物依序加入2μL SAP反应液。
SAP反应程序设置如下表：
Figure PCTCN2020086108-appb-000066
C.延伸反应(试剂盒：Agena iPLEX pro reagent set)：
(1)配置拷贝数检测延伸引物混合液，下表为该混合液中包含的引物及其浓度：
Figure PCTCN2020086108-appb-000067
(2)延伸反应体系配置如下表：
Figure PCTCN2020086108-appb-000068
延伸反应程序设置如下表：
Figure PCTCN2020086108-appb-000069
Figure PCTCN2020086108-appb-000070
D.核酸飞行质谱平台点样分析获取数据并分析，具体如下。
a)样本内校正：处理步骤D中获得的数据，获得待测样本中的目的基因和竞争物的比值，包括SMN1 E7/竞争物、SMN2 E7/竞争物、SMN1 E8/竞争物、SMN2 E8/竞争物、HBA1/竞争物、HBA2/竞争物、HBQ/竞争物及内参DNA序列的校正用比值QC/竞争物。将SMN1 E7/竞争物、SMN2 E7/竞争物、SMN1 E8/竞争物、SMN2 E8/竞争物、HBA1/竞争物、HBA2/竞争物、HBQ/竞争物比上校正用比值QC/竞争物，获得各目的基因的初校后比值，即[目的基因和竞争物比值/校正用比值]。
b)样本间校正：同步骤a，获取正常参考样本中的各目的基因初校后比值，将步骤a中获取的待测样本中各目的基因初校后比值与正常参考样本中相应的目的基因初校后比值相比，获得各目的基因终校后比值，如[SMN1 E7初校后比值 待测样本/SMN1 E7初校后比值 正常参考样本]。
c)已知正常参考样本中目的基因拷贝数均为2，故各待测样本的目的基因拷贝数即为各目的基因终校后比值*2，如SMN1 E7拷贝数＝[SMN1 E7初校后比值 待测样本/SMN1 E7初校后比值 正常参考样本]*2。
E.通过步骤D分析数据获得各样本目的基因拷贝数如表四：
表四：SMN基因和HBA基因拷贝数的联合检测结果
Figure PCTCN2020086108-appb-000071

Claims (15)

  1. 检测目的基因拷贝数的方法，包括：
    a)设计所述目的基因的扩增和延伸引物、所述目的基因的竞争物、不存在拷贝数变异的内参基因的扩增和延伸引物，和所述内参基因的竞争物，其中所述目的基因的竞争物与所述目的基因存在至少1个核苷酸的差异并且与所述目的基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性，作为与所述目的基因的竞争性扩增模板在PCR扩增反应中同时扩增，所述目的基因的延伸引物在延伸反应中能够分别延伸所述目的基因和目的基因的竞争物的扩增产物；所述内参基因的竞争物与所述内参基因存在至少1个核苷酸的差异并且与所述内参基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性，作为所述内参基因的竞争性扩增模板在PCR扩增反应中同时扩增，所述内参基因的延伸引物能够在延伸反应中分别延伸所述内参基因和内参基因的竞争物的扩增产物；
    b)在获自怀疑具有目的基因拷贝数变异的受试者的待测DNA样本和获自正常人的正常对照DNA样本中加入步骤a)所述的扩增引物，进行PCR扩增，得到目的基因、内参基因及其各自竞争物的PCR扩增产物；
    c)在所述PCR扩增产物中加入步骤a)所述的延伸引物，进行延伸反应，得到目的基因、内参基因及其各自竞争物的延伸产物；
    d)对所述延伸产物进行基质辅助激光解吸电离飞行时间质谱，并且将得到的各个延伸产物的数据进行以下分析：
    i)样本内校正：计算待测样本中目的基因的校正用比值，即，内参基因/内参基因竞争物的比值，并且获得目的基因/目的基因竞争物的比值；用目的基因/目的基因竞争物的比值除以校正用比值，得到目的基因的初校后比值，即：目的基因与目的基因竞争物的比值/内参基因与内参基因竞争物的比值；
    ii)样本间校正：按照步骤i)的方法，计算正常对照样本中目的基因的初校后比值；将步骤i)中获取的待测样本中的目的基因初校后比值与正常对照样本中相应的目的基因初校后比值相比，获得目的基因的终校后比值，即目的基因初校后比值 待测样本/目的基因初校后比值 正常参考样本；
    iii)用待测样本目的基因终校后比值乘以2，得到待测样本中目的基因的拷贝数。
  2. 权利要求1的方法，其中所述DNA样本选自体液、血液、血清、血浆、尿、唾液、汗液、痰、精液、粘液、泪液、淋巴液、羊水、间质液、肺灌洗液、脑脊液、粪便和组织样本。
  3. 权利要求1或2的方法，其中所述目的基因是SMN1、SMN2、NAIP、HBA1、HBA2和/或HBQ基因。
  4. 权利要求1-3的任一项的方法，其中所述目的基因的竞争物与所述目的基因存在1、2、3、4、5、6、7、8、9或10个核苷酸的差异，并且所述内参基因的竞争物与所述内参基因存在1、2、3、4、5、6、7、8、9或10个核苷酸的差异。
  5. 权利要求3的方法，其中所述目的基因的扩增和延伸引物选自i)SMN1 7号外显子和SMN2 7号外显子的扩增和延伸引物，其分别如SEQ ID NO：1-2和SEQ ID NO：6所示；ii)SMN1 8号外显子和SMN2 8号外显子的扩增和延伸引物，其分别如SEQ ID NO：3-4和SEQ ID NO：5所示；iii)NAIP基因5号外显子的扩增和延伸引物，其分别如SEQ ID NO：13-14和SEQ ID NO：15所示；iv)HBA1基因的扩增和延伸引物，其分别如SEQ ID NO：17-18和SEQ ID NO：24所示；v)HBA2基因的扩增和延伸引物，其分别如SEQ ID NO：19-20和SEQ ID NO：26所示；vi)HBQ基因的扩增和延伸引物，其分别如SEQ ID NO：21-22和SEQ ID NO：28所示。
  6. 权利要求3的方法，其中所述目的基因的竞争物选自i)SMN1 7号外显子和SMN2 7号外显子的竞争物，其如SEQ ID NO：7所示；ii)SMN1 8号外显子和SMN2 8号外显子的竞争物，其如SEQ ID NO：8所示；iii)NAIP基因5号外显子的竞争物，其如SEQ ID NO：16所示，iv)HBA1基因的竞争物，其如SEQ ID NO：23所示，v)HBA2基因的竞争物，其如SEQ ID NO：25所示，和vi)HBQ基因的竞争物，其如SEQ ID NO：27所示。
  7. 权利要求3的方法，其中所述内参基因是NFATC3基因，所述内参基因的扩增和延伸引物分别如SEQ ID NO：9-10和11所示，所述内参基因的竞争物如SEQ ID NO：12所示。
  8. 检测目的基因拷贝数的试剂盒，其中含有所述目的基因的扩增 和延伸引物、所述目的基因的竞争物、不存在拷贝数变异的内参基因的扩增和延伸引物，和所述内参基因的竞争物，其中所述目的基因的竞争物与所述目的基因存在至少1个核苷酸的差异并且与所述目的基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性，所述内参基因的竞争物与所述内参基因存在至少1个核苷酸的差异并且与所述内参基因具有至少50％，优选至少60％，更优选至少70％，更优选至少80％，更优选至少90％，更优选至少95％，最优选至少99％的序列同一性。
  9. 权利要求8的试剂盒，其中所述目的基因是SMN1、SMN2、NAIP、HBA1、HBA2和/或HBQ。
  10. 权利要求8或9的试剂盒，其中所述目的基因的竞争物与所述目的基因存在1、2、3、4、5、6、7、8、9或10个核苷酸的差异，并且所述内参基因的竞争物与所述内参基因存在1、2、3、4、5、6、7、8、9或10个核苷酸的差异。
  11. 权利要求9的试剂盒，其中所述目的基因的扩增和延伸引物是i)SMN1 7号外显子和SMN2 7号外显子的扩增和延伸引物，其分别如SEQ ID NO：1-2和SEQ ID NO：6所示；ii)SMN1 8号外显子和SMN2 8号外显子的扩增和延伸引物，其分别如SEQ ID NO：3-4和SEQ ID NO：5所示；iii)NAIP基因5号外显子的扩增和延伸引物，其分别如SEQ ID NO：13-14和SEQ ID NO：15所示；iv)HBA1基因的扩增和延伸引物，其分别如SEQ ID NO：17-18和SEQ ID NO：24所示；v)HBA2基因的扩增和延伸引物，其分别如SEQ ID NO：19-20和SEQ ID NO：26所示；vi)HBQ基因的扩增和延伸引物，其分别如SEQ ID NO：21-22和SEQ ID NO：28所示。
  12. 权利要求11的试剂盒，其中所述所述目的基因的竞争物选自i)SMN1 7号外显子和SMN2 7号外显子的竞争物，其如SEQ ID NO：7所示；ii)SMN1 8号外显子和SMN2 8号外显子的竞争物，其如SEQ ID NO：8所示；iii)NAIP基因5号外显子的竞争物，其如SEQ ID NO：16所示，iv)HBA1基因的竞争物，其如SEQ ID NO：23所示，v)HBA2基因的竞争物，其如SEQ ID NO：25所示，和vi)HBQ基因的竞争物，其如SEQ ID NO：27所示。
  13. 权利要求9的试剂盒，其中所述内参基因是NFATC3基因，所述内参基因的扩增和延伸引物分别如SEQ ID NO：9-10和11所示，所述内参基因的竞争物如SEQ ID NO：12所示。
  14. 引物，其如选自SEQ ID NO：1-6、SEQ ID NO：9-11、SEQ ID NO：13-15、SEQ ID NO：17-22、SEQ ID NO：24、SEQ ID NO：26和SEQ ID NO：28的序列所示。
  15. 多核苷酸，其如选自SEQ ID NO：7、8、12、16、23、25和27的序列所示。
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