Primer probe and kit for detecting smn1 gene mutation

Abstract

The application provides a primer probe and kit for detecting SMN1 gene mutation. The kit comprises primers for detecting deletion mutation of SMN1 gene exons 7 and 8 respectively, and corresponding mutation detection probes and blocking probes. The application is more accurate and reliable by reducing the interference of SMN2 pseudogene. The triple amplification of SMN1 gene exons 7 and 8 and the reference Actin gene is completed in a single reaction tube by using the principle of multiplex PCR amplification, thereby improving the efficiency. Through reaction system optimization, the micro blood sample can be used for PCR amplification without purification of genomic DNA, the pretreatment is completed in 15 minutes, the whole detection is completed in about 2.5 hours, time and purification cost are saved, and the kit is suitable for high-throughput screening and detection of neonatal dried blood spots.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a primer probe and kit for detecting SMN1 gene mutation. BACKGROUND

[0002] Spinal muscular atrophy (SMA) is a disease caused by the degeneration of motor neurons in the anterior horn of the spinal cord, leading to muscle weakness and atrophy. It is an autosomal recessive genetic disease caused by gene defects, and is not uncommon in clinical practice. The incidence is about 1 / 6000-1 / 10000, and the carrier rate in the population is about 1 / 35-1 / 50, i.e. 50 ordinary people have one carrier. If both spouses are carriers, the probability of each fetus being affected is 25%, and 50% may carry the SMA pathogenic gene. The clinical manifestations of this disease vary greatly. According to the age of onset and clinical course of patients, SMA is divided into four types from severe to mild. The common feature is the degeneration of spinal cord cells, and the clinical manifestations are progressive, symmetric, and widespread flaccid paralysis and muscle atrophy mainly in the proximal limbs, with normal intelligence and sensation. SMA patients will experience muscle atrophy and weakness due to the degeneration of spinal motor neurons, leading to the gradual loss of various motor functions, and the progression of the disease will further affect swallowing, breathing, and ultimately lead to death. It is the number one genetic killer of infants under two years old and is one of the most severe rare diseases. Early detection and diagnosis, and early intervention are of great significance.

[0003] Through neonatal screening, SMA patients can be detected early, and necessary diagnostic and treatment measures can be taken in time before the appearance of obvious muscle and nervous system symptoms, which can reduce the mortality of newborns and patients and effectively improve the prognosis of children. In China, 58.96% of SMA patients develop from 7 to 18 months of age, and severe patients account for about 89% of the total number of patients; in terms of motor function, only 22.78% of SMA patients can walk.

[0004] The gene defect of SMN1 is the main cause of spinal muscular atrophy, the main pathogenic gene of SMA is survival motor neuron 1 (SMN1), about 90% of spinal muscular atrophy patients are caused by homozygous deletion of exon 7 or homozygous deletion of exon 7 and 8 of SMN1 gene, SMN gene is located on the long arm of chromosome 5, region 3 (5q13), has 9 exons (exon 1, 2a, 2b, 3-8), the full length is about 27kb, encodes an RNA-binding protein SMN of 294 amino acids, which is essential for efficient assembly of small nuclear ribonucleoprotein (snRNP) complexes. There are two highly homologous copies of SMN1 and SMN2 genes, the telomere side is called SMN1, and the centromere side is called SMN2. The difference between the two is only 5 bases at the 3' end of each, of which 2 bases are located in exons 7 and 8, and the other 3 bases are in introns 6 and 7.

[0005] Therefore, detecting the copy number of exon 7 of SMN1 gene has important significance for clinical diagnosis of SMA and screening of normal population carriers, especially early SMA screening for newborns, which will shift the prevention and control barrier and improve the prevention and control effect.

[0006] At present, the main methods for SMA gene detection in clinical practice are: restriction fragment length polymorphism polymerase chain reaction technology (PCR-RFLP), denaturing high performance liquid chromatography analysis (PCR-DHPLC), multiple ligation-dependent probe amplification technology (MLPA), real-time fluorescent quantitative PCR, high-throughput sequencing, etc. PCR-RFLP method can only detect homozygous deletion of exon 7 of SMN1 gene, and cannot detect single copy deletion carriers. PCR-DHPLC method, MLPA method and high-throughput sequencing can be used to detect homozygous deletion type patients and heterozygous deletion type carriers, but the operation is complex, the throughput is low, the time is long, special instruments are needed, and the price is high.

[0007] The existing real-time fluorescent quantitative PCR technology detects exon 7 and 8 of SMN1 gene, mainly designs blocking primers or probes for specific sites of SMN1, but general blocking primers still cannot exclude the non-specific amplification of pseudogene SMN2, resulting in non-specific results.

[0008] The existing spinal muscular atrophy gene detection kit is mostly for purified genomic DNA, which requires more blood samples, and there is no kit for rapid detection of dry blood spot samples for newborn disease screening.

[0009] Therefore, it is urgent to develop a specific, reliable, simple and economical detection method and kit suitable for dry blood spots and other trace samples for detecting the copy number deletion of SMN1 and screening newborns with spinal muscular atrophy. SUMMARY

[0010] The present application aims at the defects of SMN2 pseudogene interference, more blood samples, complicated and time-consuming pretreatment and detection operation, high detection cost, easy pollution and low throughput in the prior art, and provides a spinal muscular atrophy gene detection kit based on fluorescence quantitative PCR technology and a preparation method thereof, which realizes newborn screening of spinal muscular atrophy by intuitively, accurately, quickly and simply detecting the copy numbers of SMN1 exons 7 and 8.

[0011] In order to achieve the object of the present application, in view of the problems existing in the real-time fluorescence quantitative PCR and the interference of SMN2 gene, two pairs of specific primers are designed for SMN1 gene by optimizing the primers of the target gene SMN1 and using the AS-PCR design principle, the upstream and downstream primers are designed for the specific sites of c.840 and INS7+215, and the difference sites are modified by LAN locked nucleic acid, so as to improve the Tm value difference caused by the difference sites of SMN1 and SMN2, improve the specificity and amplification efficiency of the primers. Meanwhile, a blocking probe is designed for the pseudogene SMN2 at the INS7+215 site and the exon 8 difference site *239, the difference site is modified by LAN locked nucleic acid, the blocking probe is completely combined with SMN2, and because SMN1 cannot be completely combined at the difference site, the Tm value is significantly reduced, so as to block the SMN2 gene without affecting the amplification of SMN1 gene. Figure 1 )。

[0012] In the first aspect, the present application provides a primer set for detecting SMN1 gene mutation, which comprises primer pair SMN1-E7-F1 and SMN1-E7-R1 (SEQ ID NO: 1-2) for detecting SMN1 gene exon 7 deletion mutation, and primer pair SMN1-E8-F1 and SMN1-E8-R1 (SEQ ID NO: 5-6) for detecting SMN1 gene exon 8 deletion mutation; the nucleotide sequences thereof are as follows:

[0013] SMN1-E7-F1: 5'-TTTATTTTCCTTACAGGGTTT C -3';

[0014] SMN1-E7-R1: 5'-GTGAAAGTATGTTTCTTCCACG T A-3';

[0015] SMN1-E8-F1: 5'-CCATCTGTAAAAGACTT G G-3';

[0016] SMN1-E8-R1: 5'-CCACATTCAAATTTTCTCAA-3';

[0017] wherein the base underlined is a locked nucleic acid (LAN) modification.

[0018] In a second aspect, the present application provides a probe used in conjunction with the primer set, including a mutation detection probe and a blocking probe.

[0019] The mutation detection probe corresponding to the exon 7 deletion mutation is SMN1-E7-P1: 5'-FAM-AAGAAGGAAGGTGCTCACAT-MGB-3'(SEQ ID NO: 3)

[0020] The blocking probe is SMN2-FB1: 5'-GTTTCTTCCAC AC AACCAAC-3'(SEQ ID NO: 4)

[0021] wherein the base underlined is a locked nucleic acid modification.

[0022] The mutation detection probe corresponding to the exon 8 deletion mutation is SMN1-E8-P1: 5'-CY5-CTCACCACCGTGCTGGCCTC-MGB-3'(SEQ ID NO: 7)

[0023] The blocking probe is SMN2-FB2: 5'-TAAAAGACT GA GGTGGGGGT-3'(SEQ ID NO: 8), wherein the base underlined is a locked nucleic acid modification.

[0024] In a third aspect, the present application provides a SMN1 gene mutation site detection kit, including a pretreatment solution, an amplification reaction solution and a mutation detection solution.

[0025] The pretreatment solution is 2-10% Chelex-100, 0-1% Tween-20 and 0-20mM NaOH; preferably 5% Chelex-100, 1% Tween-20 and 20mM NaOH.

[0026] The amplification reaction solution is: 1-3 U of hot start Taq enzyme, 0.1-0.3 U / ul of UNG enzyme, 4-10 nmol of dNTP, 4-10 nmol of dUTP, 5x PCR buffer and amplification optimization agent; wherein the amplification optimization agent is 0.1-0.5% BSA+0.02-0.2 M betaine; preferably 2 U of hot start Taq enzyme, 0.2 U of UNG enzyme, 10 nmol of dNTP, 10 nmol of dUTP, 5x PCR buffer and amplification optimization agent; wherein the amplification optimization agent is 0.2% BSA+0.2 M betaine.

[0027] The mutation detection solution (12.5 μl) is: SMN1-E7-F1 0.8 μM, SMN1-E7-R1 0.8 μM, SMN1-E8-F10.8 μM, SMN1-E8-R1 0.8 μM, Actin-F 0.4 μM, Actin-R 0.4 μM, SMN1-E7-P1 0.4 μM, SMN1-E8-P1 0.4 μM, Actin-P 0.4 μM, SMN2-FB1 0.16 μM and SMN2-FB2 0.16 μM;

[0028] Wherein, the Actin-F and the Actin-R are a pair of primers of an amplification internal reference gene Actin and a detection probe Actin-P, and the nucleotide sequences thereof are as follows:

[0029] Actin-F: 5'-AATGAGCTGCGTGTGGCT-3'

[0030] Actin-R: 5'-CAACACTGTCTTAGACACCTAGTC-3'

[0031] Actin-P: 5'-HEX-ACCCAGGTGAGTGGCCCGCTA-BHQ1-3'.

[0032] In a fourth aspect, the present application provides the use of the kit in the detection of SMN1 gene mutation.

[0033] By means of the above technical solution, the present application has at least the following advantages and beneficial effects:

[0034] (I) The present application is directed to the shortcomings of the existing spinal muscular atrophy detection reagent which requires more blood samples and purifies genomic DNA, and is not suitable for application in newborn dry blood spot screening detection, etc. The present application provides one-step processing of trace blood samples (3 ul of blood or 3 mm of dry blood spots), and the processing solution is directly subjected to subsequent amplification detection without the need for cumbersome steps such as genomic DNA purification, thereby saving cost and time and being suitable for carrier screening and newborn dry blood spot screening and detection.

[0035] (ii) By reducing the SMN2 pseudogene interference, more accurate and reliable detection. Using the principle of multiplex PCR amplification, the triple amplification of SMN1 gene exons 7 and 8 and the reference Actin gene is completed in one reaction system (single reaction tube), improving the efficiency.

[0036] (iii) Designing a double-specific primer and a blocking probe effectively reduces the SMN2 pseudogene interference, improves the detection accuracy and reliability.

[0037] (iv) Through reaction system optimization, micro blood samples can be purified for PCR amplification, 15 minutes for pretreatment, and the entire detection is completed in about 2.5 hours, saving time and purification cost, suitable for high-throughput screening and detection of neonatal dried blood spots. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The figure is a schematic diagram of the principle of SMN1 gene mutation detection of the present application.

[0039] Figure 2 The figure is a normal dried blood spot sample detected by the experimental group in the preferred embodiment of the present application.

[0040] Figure 3 The figure is a normal dried blood spot sample detected by control group 1 in the preferred embodiment of the present application.

[0041] Figure 4 The figure is a normal dried blood spot sample detected by control group 2 in the preferred embodiment of the present application.

[0042] Figure 5 The figure is a SMN1 carrier dried blood spot sample detected by the experimental group in the preferred embodiment of the present application.

[0043] Figure 6 The figure is a SMN1 carrier dried blood spot sample detected by control group 1 in the preferred embodiment of the present application.

[0044] Figure 7 The figure is a SMN1 carrier dried blood spot sample detected by control group 2 in the preferred embodiment of the present application.

[0045] Figure 8 The figure is a SMN1 exon 7 and 8 completely deleted dried blood spot sample detected by the experimental group in the preferred embodiment of the present application.

[0046] Figure 9 The figure is a SMN1 exon 7 and 8 completely deleted dried blood spot sample detected by the experimental group in the preferred embodiment of the present application.

[0047] Figure 10 The figure is a SMN1 exon 7 and 8 completely deleted dried blood spot sample detected by control group 1 in the preferred embodiment of the present application.

[0048] Figure 11For detecting the complete deletion of SMN1 exon 7 and 8 in dried blood spot samples of control group 1 in the preferred embodiment of the present application.

[0049] Figure 12 For detecting the complete deletion of SMN1 exon 7 and 8 in dried blood spot samples of control group 2 in the preferred embodiment of the present application.

[0050] Figure 13 For detecting the complete deletion of SMN1 exon 7 and 8 in dried blood spot samples of control group 2 in the preferred embodiment of the present application.

[0051] Figure 14 For detecting the complete deletion of SMN1 exon 7 and 8 in dried blood spot samples of control group 2 in the preferred embodiment of the present application. DETAILED DESCRIPTION

[0052] The following examples are intended to illustrate the present application but not to limit the scope of the present application. If not specifically indicated, the examples are carried out according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (2001) or the conditions suggested by the manufacturer's instructions.

[0053] In the present application, the nucleotide sequence of SMN1 gene in the normal human genome DNA can refer to the 5001-33072 bases of Genbank accession number NG_008691.1.

[0054] If not specifically indicated, each reagent used in the following examples is purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0055] Example 1 SMN1 gene mutation site detection reagent

[0056] The spinal muscular atrophy gene detection reagent is composed of a sample pretreatment solution, an amplification reaction solution and a mutation detection solution.

[0057] The sample pretreatment solution comprises 5% Chelex-100, 1% Tween-20 and 20 mM NaOH. Chelex-100 is purchased from Bole Life Medicine Products (Shanghai) Co., Ltd.

[0058] The amplification reaction solution comprises 2 U hot-start Taq enzyme, 0.2 U UNG enzyme, 10 nmol dNTP, 10 nmol dUTP, 5x PCR buffer and amplification optimization agent (BSA 0.2% + betaine 0.2 M) in each amplification reaction.

[0059] The mutation reaction solution comprises:

[0060] Primer SMN1-E7-F1 and SMN1-E7-R1 amplify exon 7 of SMN1, underlined is locked nucleic acid modification, probe: SMN1-E7-P1 is labeled with fluorescent group FAM and quencher group MGB, blocking probe SMN2-FB1, underlined AC base is locked nucleic acid modification, sequence is:

[0061] Primer SMN1-E7-F1: 5'-TTTATTTTCCTTACAGGGTTT C -3'

[0062] Primer SMN1-E7-R1: 5'-GTGAAAGTATGTTTCTTCCACG T A-3'

[0063] In addition, control primers without locked nucleic acid modification are designed respectively:

[0064] Primer SMN1-E7-F2: 5'-TTTATTTTCCTTACAGGGTTTC-3'

[0065] Primer SMN1-E7-R2: 5'-GTGAAAGTATGTTTCTTCCACGTA-3'

[0066] Probe SMN1-E7-P1: 5'-AAGAAGGAAGGTGCTCACAT-3' (5' end FAM, 3' end MGB modification).

[0067] Blocking probe SMN2-FB1: 5'-GTTTCTTCCAC AC AACCAAC-3'

[0068] Primer SMN1-E8-F1 and SMN1-E8-R1 amplify exon 8 of SMN1, underlined is locked nucleic acid modification, probe: SMN1-E8-P1 is labeled with fluorescent group Cy5 and quencher group MGB, blocking probe SMN2-FB2, underlined GA base is locked nucleic acid modification, sequence is:

[0069] Primer SMN1-E8-F1: 5'-CCATCTGTAAAAGACTT G G-3'

[0070] Primer SMN1-E8-R1: 5'-CCACATTCAAATTTTCTCAA-3'

[0071] In addition, control primers without locked nucleic acid modification are designed respectively:

[0072] Primer SMN1-E8-F2: 5'-CCATCTGTAAAAGACTTGG-3'

[0073] The downstream primer uses primer SMN1-E8-R1.

[0074] Probe SMN1-E8-P1: 5'-CTCACCACCGTGCTGGCCTC-3' (5' end CY5, 3' end MGB modification).

[0075] Blocking probe SMN2-FB2: 5'-TAAAAGACT GA GGTGGGGGT-3'.

[0076] Primer Actin-F and Actin-R amplify the internal reference Actin, and probe Actin-P is labeled with fluorescent gene HEX and quencher BHQ1.

[0077] Primer Actin-F: 5'-AATGAGCTGCGTGTGGCT-3'

[0078] Primer Actin-R: 5'-CAACACTGTCTTAGACACCTAGTC-3'

[0079] Probe Actin-P: 5'-ACCCAGGTGAGTGGCCCGCTA-3' (5' end HEX, 3' end BHQ1 modification).

[0080] Example 2 Detection of spinal muscular atrophy gene

[0081] Each amplification reaction solution is composed of a solvent and a solute, the solvent being water, and the solute and its concentration being: 2U hot-start Taq enzyme, 0.2U UNG enzyme, dNTP 10 nmol, dUTP 10 nmol, 5x PCR buffer and amplification optimization agent (BSA 0.2% + betaine 0.2M), which are purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0082] The amount of amplification reaction solution (1 person) is shown in Table 1.

[0083] Table 1 Amount of amplification reaction solution

[0084] Reagent 1 serving (ul) 5x PCR buffer 5 5U / ul Hot-Taq enzyme (Shanghai Sangon) 0.4 10mM dNTP (Shanghai Sangon) 1 100mM dUTP (Shanghai Sangon) 0.1 5U / ul UDG enzyme (Shanghai Sangon) 0.05 BSA (5%) 1ul Betaine (5M) 1ul Total 8.55

[0085] The mutation detection solution is composed of a solvent and a solute, the solvent being water, and the solute and its concentration being shown in Table 2.

[0086] Table 2 Solute, concentration and amount of mutation detection solution

[0087]

[0088] The detection method of SMN1 gene mutation is as follows:

[0089] The spinal muscular atrophy gene detection reagent can be used for clinical detection of spinal muscular atrophy gene SMN1, in particular, screening of spinal muscular atrophy carriers and newborn gene screening detection, and the specific operation steps of the application are as follows:

[0090] 1. Sample pretreatment:

[0091] Add 100 ul of pretreatment solution to 3 ul of whole blood or 3 mm of dry blood spots, incubate at 95°C for 10 min, then centrifuge at 5000 rpm for 5 min, and wait for use.

[0092] 2. Configuration of detection reaction:

[0093] Step 1: Take 2 ul of the extraction solution and mix it with 8.55 ul of amplification reaction solution and 12.5 ul of mutation detection solution to add to the detection reaction tube, add 1.95 ul of deionized water, the total volume is 25 ul, and centrifuge to remove bubbles.

[0094] 3. Detection

[0095] Put the detection reaction tube into a fluorescence quantitative PCR instrument, and the PCR amplification program is as follows:

[0096] 50°C for 2 min, 95°C for 10 min; 95°C for 20 s, 58°C for 50 s (collect FAM, HEX, CY5 fluorescence signals), 50 cycles;

[0097] 4. Result analysis:

[0098] Use the ΔΔCt value method for relative quantitative analysis, calculate the ΔΔCt of the detection sample = (detection sample SMN1 Ct - detection sample internal reference Ct) - (normal control sample SMN1 Ct - normal control sample internal reference Ct), and determine the ΔΔCt value range through single copy and double copy deletion control samples.

[0099] Example 3: Verification and detection effect of clinical samples

[0100] In order to test the blocking effect of the blocking probe on the SMN2 gene and reduce the interference of SMN2 on SMN1, normal samples (SMN1 copy number is 2), SMN1 carrier samples (SMN1 copy number is 1) and SMN1 deletion samples with only SMN2 gene (SMN1 copy number is 0) are selected, and the anticoagulated blood samples are made into dry blood spots, and the amplification reaction system is divided into experimental group and control group 1 and control group 2, the experimental group prepares the amplification system according to the mutation detection solution in Example 2, the experimental group uses the SMN1 gene mutation detection reagent prepared in Example 1 to detect the sample to be tested, the upstream and downstream primers SMN1-E7-F1 and SMN1-E7-R1 in the experimental group are designed for specific sites c.840 and INS7+215 respectively, and the blocking probe SMN2-FB1 is designed for the INS7+215 site.

[0101] The control group 1 is designed to amplify SMN1 exon 7 at the specific base without LAN modification, respectively for the c.840 and INS7+215 specific sites, without locked nucleic acid modification:

[0102] Primer SMN1-E7-F2: 5'-TTTATTTTCCTTACAGGGTTTC-3'

[0103] Primer SMN1-E7-R2: 5'-GTGAAAGTATGTTTCTTCCACGTA-3'

[0104] The control primer without locked nucleic acid modification is designed to amplify SMN1 exon 8:

[0105] Primer SMN1-E8-F2: 5'-CCATCTGTAAAAGACTTGG-3'

[0106] Primer SMN1-E8-R1: 5'-CCACATTCAAATTTTCTCAA-3'

[0107] The amplification system does not add blocking probes SMN2-E7-FB and SMN2-E8-FB, and the amplification system is shown in Table 3.

[0108] Table 3 Components of the amplification system of control group 1

[0109]

[0110]

[0111] The control group 2 uses the SMN1 gene mutation detection reagent prepared in Example 1 to amplify different regions of SMN1 exon 7 for comparison of detection effects, and the control group 2 is designed with the upstream primer SMN1-E7-F1 for c.840, the downstream primer SMN1-E7-R3 for another difference site INS7+100, and the blocking probe SMN2-FB3 for the INS7+100 site, which is completely combined with SMN2.

[0112] The primer and probe sequences are:

[0113] Primer SMN1-E7-R3: 5'-TGTTTTACATTAACCTTTCAACTC T T-3', the underlined T base is locked nucleic acid modification;

[0114] Blocking probe SMN2-FB3: 5'-TTCCTTACAGGGTTG TA GACAA-3', the underlined TA base is locked nucleic acid modification.

[0115] The composition of the amplification detection system of the control group 2 is shown in Table 4.

[0116] Table 4 Composition of the amplification system of the control group 2

[0117]

[0118] The experimental group and the control group 1 and the control group 2 were detected according to the SMN1 gene detection procedure in Example 2.

[0119] The results show that the experimental group and the control group 1 and the control group 2 can effectively distinguish normal samples and carrier samples. Figure 2-Figure 7 ).

[0120] For samples with complete deletion of SMN1 exons 7 and 8, there is a significant difference in effect. In the experimental group, using the locked nucleic acid modified primer and the blocking probe, for samples with deletion of SMN1 exons 7 and 8, SMN2 non-specific amplification is effectively inhibited, and no amplification signal appears. The difference in ΔΔCt between the carrier and the deletion sample is significant, and the discrimination degree and specificity are obviously enhanced. The blocking probe SMN2-FB1 and SMN2-FB2 have obvious blocking effect on SMN2, which can effectively prevent the interference of SMN2 on SMN1. Figure 8 , Figure 9 ).

[0121] In the detection system of the control group 1, the ordinary blocking primer amplifies the sample with deletion of SMN1 exons 7 and 8, and there is no blocking probe, and there is still a strong amplification signal, and SMN2 appears non-specific amplification. Figure 10 , Figure 11 ), the ΔΔCt value of the carrier and the deletion sample is relatively close, the specificity is not strong, and the discrimination degree is significantly reduced (Table 5), which is easy to lead to non-specific results.

[0122] The results show that in the control group 2, in the sample with deletion of both SMN1 exon 7 and exon 8, a lower amplification signal appears, which is caused by SMN2 non-specific amplification. Figure 12 , Figure 13 ), the combination of the primer and the blocking probe in the experimental group is better than that in the control group 2, and the specificity of SMN1 amplification is better, and there is no SMN2 non-specific amplification signal.

[0123] Table 5 Comparison of detection between the control group and the experimental group

[0124]

[0125] Example 4 Clinical sample detection

[0126] To test the application of the detection reagent in the present application in neonatal screening, 64 neonatal dried blood spot samples were collected, and the SMN1 gene mutation detection reagent prepared in Example 1 was used to detect and analyze the samples to be tested. The results showed that all 64 samples were successfully detected, and 1 carrier was found. Figure 14

[0127] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application. SEQUENCE LISTING <110> Beijing Ganjiang Biotechnology Co., Ltd. <120> Primer probe and kit for detecting SMN1 gene mutation <130> KHP221112598.1 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <400> 1 tttattttcc ttacagggtt tc 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <400> 2 gtgaaagtat gtttcttcca cgta 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 aagaaggaag gtgctcacat 20 <210> 4 <211> 20 <212> DNA ​<213> Artificial Sequence <400> 4 gtttcttcca cacaaccaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <400> 5 ccatctgtaa aagacttgg 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 ccacattcaa attttctcaa 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 ctcaccaccg tgctggcctc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 taaaagactg aggtgggggt 20

Claims

1. A primer set and probe combination for detecting deletion mutations in exons 7 and 8 of the SMN1 gene, characterized in that, The primer set includes primer pairs SMN1-E7-F1 and SMN1-E7-R1 for detecting exon 7 deletion mutations in the SMN1 gene, and primer pairs SMN1-E8-F1 and SMN1-E8-R1 for detecting exon 8 deletion mutations in the SMN1 gene; their nucleotide sequences are as follows: SMN1-E7-F1: 5′-TTTATTTTCCTTACAGGGTTT C -3′; SMN1-E7-R1: 5′-GTGAAAGTATGTTTCTTCCACG T A-3′; SMN1-E8-F1:5′-CCATCTGTAAAAGACTT G G-3′; SMN1-E8-R1: 5′-CCACATTCAAATTTTCTCAA-3′; The underlined bases are locked nucleic acid modifications; The probe combination includes mutation detection probes and blocking probes; The mutation detection probe SMN1-E7-P1 corresponding to the exon 7 deletion mutation: 5′-FAM-AAGAAGGAAGGTGCTCACAT-MGB-3′ Blocking probe: SMN2-FB1: 5′-GTTTCTTCCAC AC AACCAAC-3′ The underlined bases are locked nucleic acid modifications; The mutation detection probe SMN1-E8-P1 corresponding to the exon 8 deletion mutation is: 5′-CY5-CTCACCACCGTGCTGGCCTC-MGB-3′ Blocked probe SMN2-FB2: 5′-TAAAAGACT GA GGTGGGGGT-3′.

2. An SMN1 gene mutation detection kit, characterized in that, Includes pretreatment solution, amplification reaction solution, and mutation detection solution; The pretreatment solution is: 2-10% Chelex-100, 0-1% Tween-20, 0-20mM NaOH; The amplification reaction solution consists of: 1-3 U hot-start Taq enzyme, 0.1-0.3 U / ul UNG enzyme, 4-10 nmol dNTP, 4-10 nmoldUTP, 5×PCR buffer, and amplification optimizer; wherein the amplification optimizer is 0.1-0.5% BSA + 0.02-0.2 M betaine. The mutation detection solutions are: SMN1-E7-F1 0.8μM, SMN1-E7-R1 0.8μM, SMN1-E8-F1 0.8μM, SMN1-E8-R1 0.8μM, Actin-F 0.4μM, Actin-R 0.4μM, SMN1-E7-P1 0.4μM, SMN1-E8-P1 0.4μM, Actin-P 0.4μM, SMN2-FB1 0.16μM, and SMN2-FB2 0.16μM; The definitions of SMN1-E7-F1, SMN1-E7-R1, SMN1-E8-F1, SMN1-E8-R1, SMN1-E7-P1, SMN1-E8-P1, SMN2-FB1 and SMN2-FB2 are the same as those in claim 1; Actin-F and Actin-R are a pair of primers for amplifying the internal reference gene Actin and the detection probe Actin-P. Their nucleotide sequences are as follows: Actin-F: 5′-AATGAGCTGCGTGTGGCT-3′ Actin-R: 5′-CAACACTGCTTAGACACCTAGTC-3′ Actin-P: 5′-HEX-ACCCAGGTGAGTGGCCCGCTA-BHQ1-3′.

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