Primer probe combination and kit for detecting genotype of SMA carrier based on chromosome level and application of primer probe combination and kit

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a primer probe combination and a kit for detecting genotypes of SMA carriers based on a chromosome level and application of the primer probe combination and the kit. Wherein the primer probe combination comprises a primer probe group for detecting an exon 7 of an SMN1 gene, a probe for detecting an exon 7 of an SMN2 gene, a primer probe group for detecting a short-arm reference gene of a chromosome 5, a primer probe group for detecting a long-arm reference gene of the chromosome 5 and a primer probe group for detecting a reference gene of a human genome. The primer probe combination can eliminate chromosome copy number variation interference, greatly improves the specificity of SMA carrier genotype detection, can simultaneously detect the type I and type II of the SMA carrier genotype, has the detection sensitivity of 99%, and is suitable for large-scale population screening.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a primer-probe combination, a kit and applications thereof for detecting the genotype of SMA carriers at the chromosome level. Background Art

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by the pathogenic gene SMN1 (NG_008691.1). SMN1 (NG_008691.1) is located at 5q13.2, with transcript number NM_000344.3. The full-length SMN protein (NP_000335.1) encoded by SMN1 is ubiquitously expressed in various tissues and cells, participating in the assembly of the spliceosomal protein complex and acting as a housekeeping protein essential for eukaryotic cell survival. Biallelic pathogenic variants in SMN1 often lead to SMA. The SMN1 causative gene and the modifier gene SMN2 (OMIM 601627) are highly homologous. SMN1 determines disease onset, while SMN2 influences disease severity and progression, making the genetic diagnosis of SMA distinct from that of most other single-gene disorders. The SMN1 and SMN2 genes are a pair of highly homologous genes linearly arranged on chromosome 5, sharing 99% nucleotide identity and differing by only eight bases. A C / T at position 6, c.840, in exon 7 results in alternative splicing of exon 7 in 90% of SMN2 mRNA, resulting in only 10% of SMN2 expressing full-length, functional SMN protein.

[0003] Although existing screening technologies can detect the total copy number of the SMN1 gene in SMA carriers through nucleic acid testing, they cannot distinguish the copy distribution characteristics at the chromosome level. According to the chromosome distribution characteristics: SMA chromosome type I is a single chromosome carrying the SMN1 gene while the other is completely missing (such as the SMA genotype is 1+0 type, 2+0 type), and SMA chromosome type II is the presence of the SMN1 gene on both chromosomes (such as the SMA genotype is 1+1 type, 2+1 type). Due to technical limitations, the current mainstream screening method can only identify type I (1+0 type) with a total copy number of 1, while type I (such as 2+0 type) with a total copy number ≥ 2 is often misjudged as type II due to the inability to confirm the chromosome distribution characteristics, resulting in a missed detection rate of up to 60%. Existing detection technologies rely on complex operating procedures or high-cost sequencing, and during the sample processing process, factors such as insufficient cell integrity and amplification inhibition often affect chromosome structure analysis, further reducing the accuracy of typing. Therefore, there is an urgent need to study methods to accurately type the genotype of SMA carriers. Summary of the Invention

[0004] Aiming at the problems and deficiencies in the prior art, the purpose of the present invention is to provide a primer-probe combination, a kit and their applications for detecting the genotype of SMA carriers at the chromosomal level.

[0005] To achieve the purpose of the invention, the technical solutions adopted by the present invention are as follows:

[0006] The first aspect of the present invention provides a primer-probe combination for detecting the genotype of SMA carriers at the chromosomal level. The primer-probe combination includes a primer-probe group for detecting exon 7 of the SMN1 gene, a probe for detecting exon 7 of the SMN2 gene, a primer-probe group for detecting the reference gene on the short arm of chromosome 5, a primer-probe group for detecting the reference gene on the long arm of chromosome 5, and a primer-probe group for detecting the reference gene in the human genome. Among them, the primer-probe group for detecting exon 7 of the SMN1 gene contains an upstream primer SMN-840-F, a downstream primer SMN-840-R and a probe SMN-840C-P, and the specific nucleotide sequences are as follows:

[0007] Upstream primer SMN-840-F: 5’-TTTTTAACATCCATATAAAGCTATCTATATA-3’ (Sequence 1),

[0008] Downstream primer SMN-840-R: 5’-GAATGTGAGCACCTTCCTTC-3’ (Sequence 2),

[0009] Probe SMN-840C-P: 5’-TTTGTCTGAAACCCT-3’ (Sequence 3);

[0010] The probe for detecting exon 7 of the SMN2 gene is probe SMN-840T-P, and the nucleotide sequence is as follows:

[0011] Probe SMN-840T-P: 5’-ATTTTGTCTAAAACCC-3’ (Sequence 4).

[0012] According to the above primer-probe combination, preferably, the reference gene on the short arm of chromosome 5 is TERT, the reference gene on the long arm of chromosome 5 is MEIKIN, and the reference gene in the human genome is SDHAP1.

[0013] According to the above primer-probe combination, preferably, the primer-probe group for detecting TERT contains an upstream primer TERT-F, a downstream primer TERT-R and a probe TERT-P, and their nucleotide sequences are as follows:

[0014] Upstream primer TERT-F: 5’-GTTTCACGTGTGCTGATTTC-3’ (Sequence 5),

[0015] Downstream primer TERT-R: 5’-GCAAGGAGGCCATCCATAAA-3’ (Sequence 6),

[0016] Probe TERT-P: 5’-GTTTCCTGCGTAATTGGTGTCTGC-3’ (Sequence 7).

[0017] According to the above primer-probe combinations, preferably, the primer-probe set for detecting MEIKIN contains upstream primer MEIKIN-F, downstream primer MEIKIN-R, and probe MEIKIN-P, and their nucleotide sequences are as follows:

[0018] Upstream primer MEIKIN-F: 5’-TGTTGGCTTGTTCCCCATAG-3’ (Sequence 8),

[0019] Downstream primer MEIKIN-R: 5’-ATTTCTTTCTACCAGGAACAC-3’ (Sequence 9),

[0020] Probe MEIKIN-P: 5’-AGGTGGTTGCTAAACTCTAGGTG-3’ (Sequence 10).

[0021] According to the above primer-probe combinations, preferably, the primer-probe set for detecting SDHAP1 contains upstream primer SDHAP1-F, downstream primer SDHAP1-R, and probe SDHAP1-P, and their nucleotide sequences are as follows:

[0022] Upstream primer SDHAP1-F: 5’-ATGTGCCCTGAGCATCGAA-3’ (Sequence 11),

[0023] Downstream primer SDHAP1-R: 5’-ATTCTTCCCCAGCGTTTG-3’ (Sequence 12),

[0024] Probe SDHAP1-P: 5’-AGGCCTGGAGATAAAGTCCCTC-3’ (Sequence 13).

[0025] According to the above primer-probe combinations, preferably, the probes SMN-840C-P, SMN-840T-P, TERT-P, MEIKIN-P, and SDHAP1-P are all provided with a fluorescent modification group and a fluorescence quenching group; the fluorescent modification groups on the probes SMN-840C-P, SMN-840T-P, TERT-P, MEIKIN-P, and SDHAP1-P are all different.

[0026] According to the primer-probe combination described above, preferably, the fluorescent modification group is selected from any one of FAM, HEX, ROX, Cy5, Q705, FITC, Cy3, VIC, NED, Cy5.5, and the fluorescent quenching group is selected from any one of MGB, BHQ-1, BHQ-2, BHQ-3.

[0027] The second aspect of the present invention provides the application of the primer-probe composition described in the first aspect above in the preparation of a product for detecting the genotype of SMA carriers or for simultaneously quantitatively detecting the copy numbers of SMN1, TERT, MEIKIN, and SDHAP1.

[0028] According to the application described above, preferably, the product is a detection reagent or kit.

[0029] The third aspect of the present invention provides a kit, which contains the primer-probe composition described in the first aspect above.

[0030] According to the kit described above, preferably, the kit further includes PCR amplification reagents. More preferably, the PCR amplification reagents include: PCR buffer and DNA (Taq) polymerase.

[0031] The fourth aspect of the present invention provides a method for simultaneously quantitatively detecting the copy numbers of SMN1, TERT, MEIKIN, and SDHAP1 for non-disease diagnosis purposes, including the following steps:

[0032] (1) Extract the chromosomes of the sample;

[0033] (2) Using the chromosomes extracted in step (1) as a template, perform digital PCR amplification reaction with the primer-probe combination described in the first aspect above;

[0034] (3) After the digital PCR amplification reaction is completed, make a judgment according to the fluorescence signal.

[0035] According to the method described above, preferably, the final concentrations of the upstream primer SMN-840-F, downstream primer SMN-840-R, upstream primer TERT-F1, downstream primer TERT-R1, upstream primer MEIKIN-F1, downstream primer MEIKIN-R1, upstream primer SDHAP1-F1, and downstream primer SDHAP1-R1 in the primer-probe combination are all 400 nM; the final concentrations of the probes SMN-840C-P, SMN-840T-P, probe TERT-P1, probe MEIKIN-P1, and probe SDHAP1-P1 are 200 nM.

[0036] According to the above method, preferably, the reaction system for digital PCR amplification is as follows: 10 μL of PCR buffer, 0.5 μL of DNA polymerase, 0.6 μL of 20 μM upstream primer SMN-840-F, 0.3 μL of 20 μM probe SMN-840C-P, 0.3 μL of 20 μM probe SMN-840T-P

[0037] 0.3 μL, 0.6 μL of 20 μM downstream primer SMN-840-R, 0.6 μL of 20 μM upstream primer TERT-F1, 0.3 μL of 20 μM probe TERT-P1

[0038] 0.3 μL, 0.6 μL of 20 μM downstream primer TERT-R1, 0.6 μL of 20 μM upstream primer MEIKIN-F1, 0.3 μL of 20 μM probe MEIKIN-P1, 0.6 μL of 20 μM downstream primer MEIKIN-R1, 0.6 μL of 20 μM upstream primer SDHAP1-F1, 0.3 μL of 20 μM probe SDHAP1-P1, 0.6 μL of 20 μM downstream primer SDHAP1-R1, 5 μL of template, 8.2 μL of water.

[0039] According to the above method, preferably, the reaction conditions for digital PCR amplification are as follows: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 10 sec, annealing / extension at 54 °C for 40 sec, for a total of 45 cycles; cooling at 25 °C for 10 sec.

[0040] According to the above method, preferably, in step (1), the sample is blood.

[0041] According to the above method, preferably, in step (1), the specific operation for extracting the chromosomes of the sample is as follows: inoculate the blood into a culture medium, statically culture at 34 °C - 37.5 °C for 68 h - 72 h, then add colchicine dropwise to the culture medium, culture at 34 °C - 37.5 °C for 20 - 120 min, centrifuge to collect the cells; add potassium chloride solution (0.075 mol / L) to the collected cells, perform hypotonic treatment for 30 min - 60 min, then add fixative to the cells, mix well and centrifuge, discard the supernatant, collect the precipitate, resuspend the precipitate with physiological saline, and then centrifuge and discard the supernatant to obtain the chromosomes. More preferably, the concentration of the potassium chloride solution is 0.075 mol / L, and the fixative is composed of methanol and glacial acetic acid mixed in a volume ratio of 3:1.

[0042] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0043] (1) The primer-probe set for detecting exon 7 of the SMN1 gene in the primer-probe combination provided by the present invention is a specific sequence designed based on the differential sites between SMN1 and SMN2, and is obtained through sample testing, which can accurately genotype the copy numbers of exons 7 and 8 of the SMN1 and SMN2 genes.

[0044] (2) The present invention combines dPCR with chromosome technology for the first time. At the same time, the TERT gene located on the short arm of chromosome 5, the MEIKIN gene located on the long arm of chromosome 5, and the SDHAP1 gene located on chromosome 3 are selected as internal reference genes. The TERT internal reference gene and the MEIKIN internal reference gene can be used to judge whether the chromosome 5 of the extracted sample to be tested is broken. Digital PCR detection finds that the TERT internal reference gene and the MEIKIN internal reference gene are located in the same chamber, indicating that chromosome 5 is not broken, otherwise it indicates that chromosome 5 is broken; the SDHAP1 internal reference gene is used to exclude incomplete cell lysis or sample processing errors. If digital PCR detection finds that the SDHAP1 internal reference gene is located alone in a chamber, it indicates that other chromosome fragments are released, suggesting that the experimental conditions are normal. Therefore, through the design of multiple internal reference genes (MEIKIN, TERT, and SDH internal reference genes), the present invention can eliminate the interference of chromosomal copy number variation, greatly improve the specificity of SMA carrier genotype detection, can detect both type I and type II of SMA carrier genotypes at the same time, with a detection sensitivity as high as 99%, is suitable for large-scale population screening, can be widely applied to medical institutions, genetic disease screening centers and scientific research units, provides an efficient and low-cost screening plan for SMA carriers, significantly reduces the birth defect rate, and has great social benefits and market prospects. Description of the Drawings

[0045] Figure 1 Is a one-dimensional scatter plot detected by the FAM fluorescence detection channel of digital PCR;

[0046] Figure 2 Is a one-dimensional scatter plot detected by the HEX fluorescence detection channel of digital PCR;

[0047] Figure 3 Is a one-dimensional scatter plot detected by the FAM fluorescence detection channel of digital PCR for the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid using a mixed primer-probe system. Among them, from left to right, they correspond to the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid respectively;

[0048] Figure 4One-dimensional scatter plots of the HEX fluorescence detection channel for digital PCR of TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid using a mixed primer-probe system; from left to right, they correspond to TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid respectively;

[0049] Figure 5 One-dimensional scatter plots of the ROX fluorescence detection channel for digital PCR of TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid using a mixed primer-probe system; from left to right, they correspond to TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid respectively;

[0050] Figure 6 One-dimensional scatter plots of the CY5 fluorescence detection channel for digital PCR of TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid using a mixed primer-probe system; from left to right, they correspond to TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid respectively;

[0051] Figure 7 One-dimensional scatter plots of the Q705 fluorescence detection channel for digital PCR of TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid using a mixed primer-probe system; from left to right, they correspond to TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid respectively;

[0052] Figure 8 Sampling effect diagrams for digital PCR of chromosomes extracted by conventional blood culture techniques;

[0053] Figure 9 Sampling effect diagrams for digital PCR of chromosomes extracted by the method for extracting chromosomes from blood samples of the present invention;

[0054] Figure 10 One-dimensional diagrams of the FAM, HEX, ROX, CY5, and Q705 fluorescence detection channels after digital PCR amplification of blood sample 5. Detailed implementation manners

[0055] The following examples are only applicable for further elaboration of the present invention. It should be noted that all the technologies and scientific terms used in the present invention have the same meaning as those in the technical field to which the present invention belongs unless otherwise specified. For the experimental methods without specific conditions noted in the following examples, conventional techniques in this technical field are adopted, or the conditions recommended by the manufacturers are followed; for the reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0056] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0057] Example 1: Screening of Primers and Probes for SMN 1 Exon 7 and SMN 2 Exon 7

[0058] Download the SMN1 and SMN2 gene sequences from NCBI and perform sequence analysis. Since there is only one base difference between SMN1 exon 7 and SMN2 exon 7, specific probes targeting SMN1 and SMN2 exon 7 are designed respectively for these two different bases of the genes, and the same pair of primers is used for amplification of both genes. Since the GC content upstream of the 840 mutation site of SMN1 and SMN2 exon 7 is extremely low (<30%), the primer design is rather difficult; the amplified fragment lengths of the primer combinations given by Primer_Blast are all greater than 130bp. Therefore, the upstream primer and downstream primer are manually adjusted according to the Tm value, primer dimer structure and non-specific binding risk to obtain the candidate primer-probe sequences, and the candidate primer-probe sequences are specifically shown in Table 1.

[0059] Table 1 Candidate Primers and Probes for SMN 1 Exon 7 and SMN 2 Exon 7

[0060]

[0061]

[0062] In order to verify the effect of the candidate primers and probes designed in Table 1 for digital PCR detection, the candidate primers and probes designed in Table 1 are used for digital PCR to amplify and detect SMN 1 exon 7 and SMN 2 exon 7.

[0063] Among them, the reaction system of digital PCR is shown in Table 2 below:

[0064] Table 2 Digital PCR Reaction System

[0065] Component Volume (μL) PCR buffer 10 μL Taq polymerase 0.5 μL 20 μM Forward primer 0.6 μL 20 μM Reverse primer 0.6 μL 20 μM Probe 0.3 μL Template (human genomic DNA) 5 μL Nuclease-free water 13 μL Total volume 30 μL

[0066] Use a mature digital PCR brand on the market and perform digital PCR detection according to their respective operating procedures. Taking the fully automatic digital PCR instrument SCIDigital PCR of Shanghai Xiaohai Turtle Technology Co., Ltd. as an example, complete the digital PCR operation according to its operation manual, which specifically includes: 1) Initialization: After the device is powered on, open the "SCIDigital" software shortcut on the computer desktop, and the software will automatically connect to the device and complete the device initialization; 2) Place consumables: Open the device chamber door and correctly place the unused digital PCR chip (Shanghai Xiaohai Turtle Technology, D020-01), 12-well strip tube filled with PCR reaction solution, oil phase A and oil phase B, sampling pipette tip, and waste box in the corresponding workstations of the device in sequence; 3) Set the reaction program in the digital PCR instrument software setting interface (taking the fully automatic digital PCR instrument SCIDigital PCR of Shanghai Xiaohai Turtle Technology Co., Ltd. as an example, the set reaction program is shown in Table 3 below); 4) Run the experiment, wait for the program to end, and view the analysis data; The analysis software of each digital PCR platform is different. Here, taking the analysis software supporting the fully automatic digital PCR instrument SCIDigital PCR of Shanghai Xiaohai Turtle Technology Co., Ltd. as an example, select the FAM and HEX fluorescence detection channels to analyze its one-dimensional scatter plot and copy number.

[0067] Table 3 shows the reaction program set in the digital PCR instrument software setting interface

[0068]

[0069] The detection results of the FAM and HEX fluorescence detection channels of digital PCR are as Figure 1 and Figure 2 shown.

[0070] Figure 1 In [relevant figure], a and b are one-dimensional scatter plots detected by the FAM fluorescence detection channel for digital PCR detection of exon 7 of SMN 1 and exon 7 of SMN 2 using the candidate primer-probe combination (forward primer SMN-840-F1, reverse primer SMN-840-R1, probe SMN-840C-P1, and probe SMN-840T-P1); Figure 1 In [relevant figure], c and d are one-dimensional scatter plots detected by the FAM fluorescence detection channel for digital PCR detection of exon 7 of SMN1 and exon 7 of SMN 2 using the candidate primer-probe combination (forward primer SMN-840-F2, reverse primer SMN-840-R2, probe SMN-840C-P2, and probe SMN-840T-P3); Figure 1In e and f, they are one-dimensional scatter plots detected by the FAM fluorescence detection channel of digital PCR using the candidate primer-probe combination (upstream primer SMN-840-F2, downstream primer SMN-840-R2, probe SMN-840C-P2, and probe SMN-840T-P4) for detecting exon 7 of SMN 1 and exon 7 of SMN 2. From Figure 1 it can be seen that the combination of the upstream primer SMN-840-F2, the downstream primer SMN-840-R2, and the probe SMN-840C-P2 has the best detection signal separation for exon 7 of SMN 1.

[0071] Figure 2 In a and b, they are one-dimensional scatter plots detected by the HEX fluorescence detection channel of digital PCR using the candidate primer-probe combination (upstream primer SMN-840-F1, downstream primer SMN-840-R1, probe SMN-840C-P1, and probe SMN-840T-P1) for detecting exon 7 of SMN 1 and exon 7 of SMN 2; Figure 2 In c and d, they are one-dimensional scatter plots detected by the HEX fluorescence detection channel of digital PCR using the candidate primer-probe combination (upstream primer SMN-840-F2, downstream primer SMN-840-R2, probe SMN-840C-P2, and probe SMN-840T-P3) for detecting exon 7 of SMN1 and exon 7 of SMN 2; Figure 2 In e and f, they are one-dimensional scatter plots detected by the HEX fluorescence detection channel of digital PCR using the candidate primer-probe combination (upstream primer SMN-840-F2, downstream primer SMN-840-R2, probe SMN-840C-P2, and probe SMN-840T-P4) for detecting exon 7 of SMN 1 and exon 7 of SMN 2. From Figure 2 it can be seen that the combination of the upstream primer SMN-840-F2, the downstream primer SMN-840-R2, and the probe SMN-840T-P4 has the best detection signal separation for exon 7 of SMN 2.

[0072] Therefore, considering the detection signals of exon 7 of SMN 1 and exon 7 of SMN 2 comprehensively, the combination of the upstream primer SMN-840-F2, the downstream primer SMN-840-R2, the probe SMN-840C-P2, and the probe SMN-840T-P4 is selected as the primer-probe combination for detecting the SMN gene in the present invention. For the convenience of description below, the upstream primer SMN-840-F2 is uniformly denoted as the upstream primer SMN-840-F, the downstream primer SMN-840-R2 is uniformly denoted as the downstream primer SMN-840-R, the probe SMN-840C-P2 is uniformly denoted as the probe SMN-840C-P, and the probe SMN-840T-P4 is uniformly denoted as the probe SMN-840T-P.

[0073] Example 2: Screening of Primers and Probes for Reference Genes

[0074] In the present invention, the TERT gene located on the short arm of chromosome 5 is selected as reference gene 1, the MEIKIN gene located on the long arm of chromosome 5 is selected as reference gene 2, and the SDHAP1 gene located on chromosome 3 is selected as reference gene 3. Among them, the TERT reference gene and the MEIKIN reference gene are used to determine whether chromosome 5 is broken. If it is found by digital PCR that the TERT reference gene and the MEIKIN reference gene are in the same chamber, it indicates that chromosome 5 is not broken; otherwise, it indicates that chromosome 5 is broken. The SDHAP1 reference gene is used to exclude incomplete cell lysis or sample processing errors. If it is found by digital PCR that the SDHAP1 reference gene is alone in a chamber, it indicates that other chromosome fragments are released, suggesting that the experimental conditions are normal.

[0075] According to the Tm value of the secondary structure, primer dimer structure and non-specific binding risk, Primer_Blast design and manual optimization are used to obtain the amplification primer and probe sequences of the TERT reference gene, MEIKIN reference gene, and SDHAP1 reference gene, as shown in Table 4 specifically.

[0076] Table 4 Amplification Primers and Probes for TERT Reference Gene, MEIKIN Reference Gene, and SDHAP1 Reference Gene

[0077]

[0078] To verify the detection effects of the three primer-probe combinations of reference genes designed in Table 4, the amplification primer-probe combinations of the above TERT reference gene, MEIKIN reference gene, and SDHAP1 reference gene are combined with the primer-probe combination for detecting exon 7 of SMN1 and exon 7 of SMN2 screened in Example 1 to form a mixed primer-probe system, and digital PCR is performed using this mixed primer-probe system to detect the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid respectively.

[0079] Among them, the reaction system of digital PCR is as shown in Table 5 below:

[0080] Table 5 Reaction System of Digital PCR

[0081]

[0082]

[0083] Adopt a mature digital PCR brand on the market and perform digital PCR detection according to their respective operation procedures. Taking the fully automatic digital PCR instrument SCIDigital PCR of Shanghai Xiaohai Turtle Technology Co., Ltd. as an example, complete the digital PCR operation according to its operation manual, specifically including: 1) Initialization: After the device is powered on, open the "SCIDigital" software shortcut on the computer desktop, and the software will automatically connect to the device and complete the device initialization; 2) Place consumables: Open the device chamber door and correctly place the unused digital PCR chip (Shanghai Xiaohai Turtle Technology, D020-01), 12-well strip tube filled with PCR reaction solution, oil phase A and oil phase B, sampling pipette tip, and waste box in the corresponding workstations of the device in sequence; 3) Set the reaction program in the software setting interface of the digital PCR instrument (taking the fully automatic digital PCR instrument SCIDigital PCR of Shanghai Xiaohai Turtle Technology Co., Ltd. as an example, the set reaction program is shown in Table 6 below); 4) Run the experiment, wait for the program to end, and view the analysis data; The analysis software of each digital PCR platform is different. Here, taking the analysis software supporting the fully automatic digital PCR instrument SCIDigital PCR of Shanghai Xiaohai Turtle Technology Co., Ltd. as an example, select the FAM, HEX, ROX, CY5, and Q705 fluorescence detection channels to analyze its one-dimensional scatter plot and copy number.

[0084] Table 6 shows the reaction program set in the software setting interface of the digital PCR instrument

[0085]

[0086] Among them, the one-dimensional scatter plots of the FAM fluorescence detection channel of digital PCR for the TERT plasmid (plasmid containing the TERT gene), MEIKIN plasmid (plasmid containing the MEIKIN gene), and SDHAP1 plasmid (plasmid containing the SDHAP1 gene) by the mixed primer-probe system are as Figure 3 shown. The one-dimensional scatter plots of the HEX fluorescence detection channel of digital PCR for the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid by the mixed primer-probe system are as Figure 4 shown. The one-dimensional scatter plots of the ROX fluorescence detection channel of digital PCR for the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid by the mixed primer-probe system are as Figure 5 shown. The one-dimensional scatter plots of the CY5 fluorescence detection channel of digital PCR for the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid by the mixed primer-probe system are as Figure 6 shown. The one-dimensional scatter plots of the Q705 fluorescence detection channel of digital PCR for the TERT plasmid, MEIKIN plasmid, and SDHAP1 plasmid by the mixed primer-probe system are as Figure 7 shown.

[0087] As can be seen from Figure 3 - Figure 7 the above, the primer-probe sets designed for detecting the TERT endogenous gene, MEIKIN endogenous gene, SDHAP1 endogenous gene and for detecting exon 7 of the SMN1 gene, and the probe combination for detecting exon 7 of the SMN2 gene can be normally amplified on their respective templates and have accurate quantification values, and there is no non-specific amplification on other templates, indicating that the mixed system has good specificity and no cross-risk, providing favorable conditions for subsequent sample detection.

[0088] Therefore, based on the above screening results, the primer-probe combination shown in Table 7 is selected as the primer-probe combination for detecting the genotype of SMA carriers at the chromosomal level in the present invention.

[0089] Table 7 Primer-probe combination for detecting the genotype of SMA carriers at the chromosomal level in the present invention

[0090]

[0091] Based on the screening of the above primer-probe combination, the present invention provides a kit, which contains the primer-probe combination for detecting the genotype of SMA carriers at the chromosomal level shown in Table 7 above, and also contains PCR amplification reagents (PCR amplification reagents include PCR buffer, DNA (Taq) polymerase, etc.). This kit can be used for detecting the genotype of SMA carriers or for quantitatively detecting the copy numbers of SMN1, TERT, MEIKIN, and SDHAP1 simultaneously.

[0092] Example 3: Sample Detection 1. Sample Selection: Twenty blood samples that have been detected for the SMN1 gene using an existing commercially available SMA fluorescence kit (Shanghai Wuseshi Medical Technology Co., Ltd., Detection Kit for Exon Deletion of Survival Motor Neuron 1 (SMN1) Gene (Fluorescence Quantitative PCR Method)) are selected. Among them, 4 are SMA carriers, and their SMN1 gene is of the 1+0 type (i.e., the chromosomal typing is type I, and the chromosomal typing of type I includes the SMN1 gene of the 1+0 type and the SMN1 gene of the 2+0 type. Since the carriers of the SMN1 gene of the 2+0 type account for a small proportion, they are not included in the experimental samples due to actual sample collection conditions); 16 are normal people, and their SMN1 gene is of the 1+1 type (i.e., the chromosomal typing is type II, and the chromosomal typing of type II includes the SMN1 gene of the 1+1 type and the SMN1 gene of the 2+1 type. Since the SMN1 gene of the 2+1 type accounts for a small proportion, it is not included in the experimental samples due to actual sample collection conditions). The results of detecting the SMN1 genotype of the 20 samples by the fluorescence PCR platform are shown in Table 8.

[0093] Table 8 Results of detecting the SMN1 genotype of 20 blood samples by the fluorescence PCR platform

[0094] Sample number Genotype Sample number Genotype Sample number Genotype Sample number Genotype Sample 1 1 + 0 type Sample 6 1 + 1 type Sample 11 1 + 1 type Sample 16 1 + 1 type Sample 2 1 + 0 type Sample 7 1 + 1 type Sample 12 1 + 1 type Sample 17 1 + 1 type Sample 3 1 + 0 type Sample 8 1 + 1 type Sample 13 1 + 1 type Sample 18 1 + 1 type Sample 4 1 + 0 type Sample 9 1 + 1 type Sample 14 1 + 1 type Sample 19 1 + 1 type Sample 5 1 + 1 type Sample 10 1 + 1 type Sample 15 1 + 1 type Sample 20 1 + 1 type

[0095] 2. Extraction of Chromosomes from Blood Samples

[0096] Chromosomes were extracted from 20 blood samples. The specific steps of the chromosome extraction method for blood samples are as follows:

[0097] (1) Preparation of culture medium:

[0098] After thawing the culture medium at room temperature (avoid repeated freezing and thawing), place it at room temperature or pre-warm it at 37°C and mix well. The thawed but unused culture medium can be stored in a 4°C refrigerator for within one week.

[0099] (2) Inoculation:

[0100] Inoculation by direct blood collection with a syringe: Remove the lid of the culture medium bottle (containing the culture medium), disinfect it by wiping with 75% alcohol. Sterilely draw 1 - 3 mL of the patient's blood with a syringe (7-gauge needle) (when drawing blood, pay attention to drawing blood after the disinfectant at the blood collection site has dried, or wipe with 75% alcohol after iodophor disinfection), directly insert it into the inner lid, inject 35 drops of blood, shake well, and place it in a 37°C incubator for culture. The remaining blood is injected into a sterile heparin sodium (lithium) anticoagulant tube (green-top tube) and stored in a 4°C refrigerator for later use.

[0101] (3) Culture:

[0102] Place the culture medium inoculated with blood directly into a 37°C constant temperature incubator and let it stand for culture for 68 - 72 hours.

[0103] (4) Treatment with colchicine (colcemid):

[0104] Directly add 4 drops of colchicine to the culture medium after the culture in step (3) with a syringe, and continue to culture at 37°C for 50 minutes. The role of colchicine (colcemid) is to break the spindle fibers and stop cell division at the metaphase to obtain chromosomes.

[0105] (5) Chromosome treatment

[0106] 1) Cell collection and hypotonic treatment: Centrifuge the culture medium after the treatment in step (4) directly at 1500 rpm for 10 min, discard the supernatant to obtain cells; add 8 mL of pre-warmed 0.075 mol / L potassium chloride solution at 37°C to the cells, pipette and mix well until there are no blood clots, and perform hypotonic treatment for 30 min;

[0107] 2) Pre-fixation: Add 0.5 mL of freshly prepared fixative (the fixative is composed of methanol and glacial acetic acid mixed in a volume ratio of 3:1) to the cells after the treatment in step 1), mix well, centrifuge at 1500 rpm for 10 min, and discard the supernatant;

[0108] 3) First fixation: Add 8 mL of fixative to the cells processed in step 2), mix well, centrifuge at 1500 rpm for 10 min, and discard the supernatant;

[0109] 4) Second fixation: Add 8 mL of fixative to the cells processed in step 3), mix well, centrifuge at 1500 rpm for 10 min, and discard the supernatant;

[0110] 5) Washing: Add 1 mL of normal saline to wash the cells processed in step 4), pipette and mix well, resuspend the precipitate in a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 10 min, and discard the supernatant to obtain chromosomes. Add 20 - 50 μL of nuclease-free water to the extracted chromosomes, pipette 10 times with a pipette gun and then store, which can be directly used as a staining solution sample for subsequent digital PCR.

[0111] During the process of extracting chromosomes from blood samples in the present invention, the cells after hypotonic treatment are fixed multiple times, which can reduce the proportion of unbroken cells and greatly improve the extraction efficiency of peripheral blood chromosomes.

[0112] To verify the influence of the method for extracting chromosomes from blood samples in the present invention on digital PCR, the method for extracting chromosomes from blood samples described in step 2 above and the conventional blood culture technique (the conventional blood culture technique is basically the same as the steps of the above "method for extracting chromosomes from blood samples", the difference is that: "(5) In chromosome treatment, after pre-fixation treatment in 2), add 8 mL of fixative to the cells processed in step 2), mix well, centrifuge at 1500 rpm for 10 min, discard the supernatant to obtain chromosomes, and store at 2 - 8 °C") are used to extract chromosomes from the same blood sample respectively. Then, the primer-probe combinations for detecting the genotype of SMA carriers at the chromosome level in Table 7 of Example 2 are used to perform digital PCR amplification detection on the chromosomes extracted by the two chromosome extraction methods. The reaction system for digital PCR amplification detection is as shown in Table 5 of Example 2, and the reaction conditions are as shown in Table 6 of Example 2. The specific operation steps of digital PCR are the same as those in Example 2.

[0113] The injection effect detection diagrams of the chromosomes extracted by the two chromosome extraction methods during digital PCR are as Figure 8 、 Figure 9 shown.

[0114] As Figure 8 can be seen, when the chromosomes extracted by the existing conventional blood culture technique are used for digital PCR injection detection, the positive points are bright and weak, and there are bright dot particles in the weak positive points, which is the result of in-situ amplification of intact unbroken cells entering the reaction chamber. As Figure 9It can be seen that when the chromosome extraction method of the present invention is used to extract chromosomes for digital PCR sample injection detection, the positive brightness of each sample is uniform and the distribution of each bright spot is uniform. These are all chromosomes released from broken cells. This shows that the blood sample chromosome extraction method provided by the present invention can effectively improve the extraction efficiency of peripheral blood chromosomes and reduce the proportion of unbroken cells.

[0115] 4. Digital PCR detection of 20 blood samples:

[0116] (1). Use the primer-probe combination for detecting the genotype of SMA carriers at the chromosome level in Table 7 of Example 2 to perform digital PCR amplification detection on the chromosomes of 20 blood samples. The reaction system for digital PCR amplification detection is as shown in Table 5 of Example 2, and the reaction conditions are as shown in Table 6 of Example 2. The specific operation steps of digital PCR are the same as those in Example 2.

[0117] Among them, taking Sample 5 as an example, after digital PCR amplification by SCIDigital PCR, the one-dimensional diagrams of the FAM, HEX, ROX, CY5, and Q705 fluorescence detection channels output by its supporting analysis software are as Figure 10 shown.

[0118] It can be Figure 10 seen that the positive and negative points in each channel are clearly clustered, indicating that the mixed system of the present invention can be normally amplified in actual samples.

[0119] Taking Sample 5 as an example, after digital PCR amplification by SCIDigital PCR, the results of the FAM, HEX, ROX, CY5, and Q705 fluorescence detection channels output by its supporting analysis software are shown in Table 9.

[0120] Table 9 Detection results of Sample 5 in FAM, HEX, ROX, CY5, and Q705 fluorescence detection channels

[0121]

[0122] (2) Digital PCR result analysis:

[0123] In the same chamber of digital PCR, ROX-CY5 co-emits light, indicating that chromosome 5 is complete but the SMN 840C gene is deleted, defined as "SMN-"; in the same chamber of digital PCR, FAM-ROX-CY5 co-emits light, indicating that chromosome 5 is complete and the SMN 840C gene is retained, defined as "SMN+"; in the same chamber of digital PCR, ROX-CY5-Q705 or FAM-ROX-CY5-Q705 co-emits light, indicating that the chromosome is complete.

[0124] The SMA genotype interpretation criteria are shown in Table 10 below.

[0125] Table 10 SMA Genotype Interpretation Criteria

[0126] Chromosome typing SMN - proportion Copy number ratio Judgment threshold Type I (carrier) SMN - / SMN + ROX - CY5 / FAM - ROX - CY5 >80％ Type II (normal person) SMN - / SMN + ROX - CY5 / FAM - ROX - CY5 ≤80％

[0127] Taking sample 5 as an example, the co-positive data output by the analysis software supporting SCIDigital PCR is shown in Table 11.

[0128] Table 11 Digital PCR Test Analysis Results of Sample 5

[0129]

[0130]

[0131] As can be seen from Table 11, the proportion of SMN copies is 6.783 / 40.408 = 16.78% (SMN1- / SMN1+), which is lower than the judgment threshold of 80%. Therefore, the SMA genotype of sample 5 is type II, belonging to normal people, which is consistent with the test results of the commercially available SMA fluorescence kit (Shanghai Wuseshi Medical Technology Co., Ltd., Detection Kit for Exon Deletion of Survival Motor Neuron Gene 1 (SMN1) (Fluorescence Quantitative PCR Method)).

[0132] The digital PCR test analysis results of 20 samples are shown in Table 12.

[0133] Table 12 Digital PCR Test Analysis Results of 20 Samples

[0134]

[0135] As can be seen from Table 12, after performing digital PCR on 20 samples using the primer-probe combination designed in the present invention for detecting the SMA carrier genotype at the chromosomal level, the consistency of the SMA genotype test results with those of the commercially available SMA fluorescence kit is 100%. This indicates that the detection system of the present invention can effectively overcome the influence of SMN 840T, which is highly homologous to SMN840C, on the quantitative detection of SMN gene copies at the chromosomal level. By combining a simple and effective chromosomal treatment method with a digital PCR platform, it realizes the effective differentiation of normal people, SMA pathogenic gene carriers, and SMA patients, providing a powerful tool for the prevention and control of SMA.

[0136] The above is the description of the embodiments of the present invention. By describing the disclosed embodiments, those skilled in the art can implement or use the present invention, rather than limiting the present invention. All the contents related to the present invention cannot be fully reflected in the above embodiments. Any equivalent changes or modifications made by any professional familiar with this technology without departing from the spirit or scope of the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A primer-probe combination for detecting the genotype of SMA carriers at the chromosomal level, characterized in that, The primer-probe combination includes a primer-probe set for detecting exon 7 of the SMN1 gene, a probe for detecting exon 7 of the SMN2 gene, a primer-probe set for detecting the reference gene on the short arm of chromosome 5, a primer-probe set for detecting the reference gene on the long arm of chromosome 5, and a primer-probe set for detecting the reference gene in the human genome; wherein, the primer-probe set for detecting exon 7 of the SMN1 gene contains an upstream primer SMN-840-F, a downstream primer SMN-840-R, and a probe SMN-840C-P, and their nucleotide sequences are as follows: Upstream primer SMN-840-F: 5’-TTTTTAACATCCATATAAAGCTATCTATATA-3’, Downstream primer SMN-840-R: 5’-GAATGTGAGCACCTTCCTTC-3’, Probe SMN-840C-P: 5’-TTTGTCTGAAACCCT-3’; The probe for detecting exon 7 of the SMN2 gene is probe SMN-840T-P, and its nucleotide sequence is as follows: Probe SMN-840T-P: 5’-ATTTTGTCTAAAACCC-3’.

2. The primer-probe combination according to claim 1, wherein The reference gene on the short arm of chromosome 5 is TERT, the reference gene on the long arm of chromosome 5 is MEIKIN, and the reference gene in the human genome is SDHAP1; The primer-probe set for detecting TERT contains an upstream primer TERT-F, a downstream primer TERT-R, and a probe TERT-P, and their nucleotide sequences are as follows: Upstream primer TERT-F: 5’-GTTTCACGTGTGCTGATTTC-3’, Downstream primer TERT-R: 5’-GCAAGGAGGCCATCCATAAA-3’, Probe TERT-P: 5’-GTTTCCTGCGTAATTGGTGTCTGC-3’; The primer-probe set for detecting MEIKIN contains an upstream primer MEIKIN-F, a downstream primer MEIKIN-R, and a probe MEIKIN-P, and their nucleotide sequences are as follows: Upstream primer MEIKIN-F: 5’-TGTTGGCTTGTTCCCCATAG-3’, Downstream primer MEIKIN-R: 5’-ATTTCTTTCTACCAGGAACAC-3’, Probe MEIKIN-P: 5’-AGGTGGTTGCTAAACTCTAGGTG-3’; The primer-probe set for detecting SDHAP1 contains an upstream primer SDHAP1-F, a downstream primer SDHAP1-R, and a probe SDHAP1-P, and their nucleotide sequences are as follows: Upstream primer SDHAP1-F: 5’-ATGTGCCCTGAGCATCGAA-3’, Downstream primer SDHAP1-R: 5’-ATTCTTCCCCAGCGTTTG-3’, Probe SDHAP1-P: 5’-AGGCCTGGAGATAAAGTCCCTC-3’.

3. The primer-probe combination according to claim 1 or 2, characterized in that, The probes SMN-840C-P, SMN-840T-P, TERT-P, MEIKIN-P, and SDHAP1-P are all provided with a fluorescent modification group and a fluorescence quenching group; the fluorescent modification groups on the probes SMN-840C-P, SMN-840T-P, TERT-P, MEIKIN-P, and SDHAP1-P are all different.

4. The primer-probe combination according to claim 3, wherein The fluorescent modification group is selected from any one of FAM, HEX, ROX, Cy5, Q705, FITC, Cy3, VIC, NED, and Cy5.5, and the fluorescence quenching group is selected from any one of MGB, BHQ-1, BHQ-2, and BHQ-3.

5. Use of the primer-probe composition according to any one of claims 2-4 in the preparation of a product for detecting the genotype of SMA carriers or for simultaneously quantitatively detecting the copy numbers of SMN1, TERT, MEIKIN, and SDHAP1.

6. A kit, characterized in that, The kit contains the primer-probe combination according to any one of claims 1-4.

7. The kit according to claim 6, wherein, The kit further includes PCR amplification reagents.

8. A method for quantitatively detecting the copy numbers of SMN1, TERT, MEIKIN, and SDHAP1 simultaneously for non-diagnostic purposes, characterized in that, Comprising the following steps: (1) Extracting the chromosomes of the sample; (2) Using the chromosomes extracted in step (1) as a template, performing digital PCR amplification reaction with the primer-probe combination according to any one of claims 2-4; (3) After the digital PCR amplification reaction is completed, making a judgment based on the fluorescence signal.

9. The method according to claim 8, characterized in that In the primer-probe combination, the final concentrations of the upstream primer SMN-840-F, downstream primer SMN-840-R, upstream primer TERT-F1, downstream primer TERT-R1, upstream primer MEIKIN-F1, downstream primer MEIKIN-R1, upstream primer SDHAP1-F1, and downstream primer SDHAP1-R1 are all 400 nM; the final concentrations of the probes SMN-840C-P, SMN-840T-P, TERT-P1, MEIKIN-P1, and SDHAP1-P1 are 200 nM.

10. The method according to claim 8 or 9, characterized in that The reaction system for digital PCR amplification is as follows: 10 μL of PCR buffer, 0.5 μL of DNA polymerase, 0.6 μL of 20 μM upstream primer SMN-840-F, 0.3 μL of 20 μM probe SMN-840C-P, 0.3 μL of 20 μM probe SMN-840T-P, 0.6 μL of 20 μM downstream primer SMN-840-R, 0.6 μL of 20 μM upstream primer TERT-F1, 0.3 μL of 20 μM probe TERT-P1, 0.6 μL of 20 μM downstream primer TERT-R1, 0.6 μL of 20 μM upstream primer MEIKIN-F1, 0.3 μL of 20 μM probe MEIKIN-P1, 0.6 μL of 20 μM downstream primer MEIKIN-R1, 0.6 μL of 20 μM upstream primer SDHAP1-F1, 0.3 μL of 20 μM probe SDHAP1-P1, 0.6 μL of 20 μM downstream primer SDHAP1-R1, 5 μL of template, and 8.2 μL of water; The reaction conditions for digital PCR amplification are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 sec, annealing / extension at 54°C for 40 sec, for a total of 45 cycles; cooling at 25°C for 10 sec.

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