SMA detection kit based on digital PCR technology

Abstract

This invention provides an SMA detection kit based on digital PCR technology. This study utilizes digital PCR detection technology to simultaneously detect the copy numbers of the SMN1 and SMN2 genes in the same reaction, and also to detect small mutations in the SMN1 gene and other genes simultaneously, improving diagnostic accuracy, simplifying the operation process, and increasing timeliness. It can be applied to SMA carrier screening, clinical diagnosis, and disease subtyping, while also saving on testing costs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an SMA detection kit based on digital PCR technology. Background Technology

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by symmetrical muscle weakness and atrophy due to degeneration of the anterior horn cells of the spinal cord. It is a common motor neuron disease. Typical manifestations include muscle weakness, hypotonia, and atrophy, resulting in limited motor function such as standing and walking. Motor development is significantly delayed compared to normal children, and patients may be unable to perform even basic life-sustaining actions such as chewing, swallowing, and breathing, but intellectual development remains normal. Clinical presentations vary greatly among patients. Based on age of onset, acquired motor function, and rate of disease progression, SMA can be classified into types I, II, III, and IV, with type I SMA exhibiting the most severe phenotype. The severity of the SMA phenotype is correlated with the copy number of the SMN2 gene.

[0003] People of any age, race, and gender can develop SMA. The incidence of SMA in newborns is 1 / 5000-1 / 10000, the prenatal carrier rate is 1 / 35-1 / 50, and the high-risk rate in families is approximately 1 / 1600.

[0004] The SMN gene is located in the 5q13 region and has two highly homologous copies: SMN1 and SMN2. The SMN1 gene expresses the full-length and stable SMN functional protein and is the determining gene for SMA (Small Infantile Mass Atrophy). Defects in the SMN1 gene cause the SMA phenotype. The SMN2 gene is highly similar to the SMN1 gene sequence, differing by only 5 bases, located in exons 7 and 8 and introns 6 and 7, respectively. The difference in the C / T ratio in exon 7 causes exon skipping during the splicing of the primary transcript pre-mRNA in SMN2, resulting in mRNA lacking exon 7 compared to SMN1. Studies have shown that only 10%-15% of the SMN2 gene can express the full-length SMN protein; therefore, it is also considered a compensatory gene for SMA. Statistics show that approximately 95% of SMA patients have homozygous deletion of the SMN1 gene (homozygous deletion of exons 7 and / or 8), while approximately 5% have compound heterozygous mutations resulting from a combination of heterozygous deletion of the SMN1 gene (heterozygous deletion of exons 7 and / or 8) and point mutations.

[0005] Based on the pathogenic gene characteristics of SMA, existing gene diagnostic technologies mainly analyze whether there are deletions and the number of deletions in exon 7 and / or exon 8 of the SMN1 gene. However, exon 8 of the SMN1 gene does not participate in SMN protein expression, so detecting the deletion of exon 8 of the SMN1 gene has little clinical significance. Currently, the main techniques used are restriction fragment length polymorphism (RFLP), multiplex ligation probe amplification (MLPA), and quantitative real-time PCR (qPCR). RFLP is simple to operate and does not require sophisticated equipment, but it can only detect homozygous deletions of exon 7 of SMN1. It first uses a specific probe to hybridize with the target sequence. The specific probe contains two oligonucleotides, and ligation can only occur when the two oligonucleotides hybridize successfully. After ligation, multiplex PCR amplification is performed to produce products of different lengths. Then, the products of different sizes are analyzed on a capillary sequencer, and the changes in peak height reflect the gene deletion. If a point mutation or deletion occurs in the primer region of the target sequence, the amplification peak of the corresponding probe will decrease or disappear, which can easily lead to false negative results. qPCR is simple and rapid, but it relies on a standard curve for quantification, and existing kits cannot simultaneously detect SMN1 exons 7 and 8 and the SMN2 gene. Whether the difference in amplification efficiency between the target gene and the internal reference gene consistently conforms to the mathematical basis of the 2-ΔΔCt relative quantification algorithm also needs verification. Therefore, the reliability of this method in carrier detection is questionable. Furthermore, neither of these two methods is suitable for detecting SMN1 gene point mutations (including missense, nonsense, frameshift, and splicing site mutations).

[0006] In addition, due to the large number of SMA carriers, most existing methods test whole blood samples, which is costly and time-consuming for nucleic acid pretreatment, making it unsuitable for large-scale screening.

[0007] Currently, there is no technical solution capable of simultaneously detecting the copy numbers, point mutations, and other genes of the SMN1 and SMN2 genes in a single tube. Therefore, the field still needs to explore a new method to simultaneously detect the copy numbers and point mutations of the SMN1 and SMN2 genes, improving diagnostic accuracy, simplifying procedures, and increasing timeliness. This method could be applied to SMA carrier screening, clinical diagnosis, and disease classification. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an SMA detection kit based on digital PCR technology to solve the problems of complex and inconvenient SMA diagnosis in the prior art.

[0009] One aspect of the present invention provides a kit for detecting the SMN1 and SMN2 genes using digital PCR technology, the kit containing at least the following probes and primers:

[0010] a. An upstream primer for detecting the 7th exon of SMN1 or SMN2, the nucleotide sequence of which is shown in SEQ ID NO.1, and a downstream primer, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0011] b. A probe for detecting exon 7 of SMN1, the nucleotide sequence of which is shown in SEQ ID NO.3;

[0012] c. A probe for detecting exon 7 of SMN2, the nucleotide sequence of which is shown in SEQ ID NO.4;

[0013] d. Any one or more of the following groups of probes and primers used to detect the target gene:

[0014]

[0015] Each of the probes described above has a pair of distinct fluorescent reporter groups and fluorescent quencher groups attached to both ends.

[0016] The technical solution of this application should be used in conjunction with digital PCR applications, and should have high specificity and sensitivity, and should also be able to detect small mutations and other genes.

[0017] All the probes and primers mentioned above can be used in a single reaction system.

[0018] Furthermore, the kit also includes primers and probes for detecting an internal reference gene. The internal reference gene can be MRPS18C. Correspondingly, the upstream primer for detecting the internal reference gene MRPS18C has the nucleotide sequence shown in SEQ ID NO. 5, the downstream primer has the nucleotide sequence shown in SEQ ID NO. 6, and the probe nucleotide sequence is shown in SEQ ID NO. 7.

[0019] Furthermore, the fluorescent group labeled at the 5' end of the probe is selected from any one of FAM, HEX, TET, ROX, CY3, CY5, CY5.5, VIC, JOE, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 647, or Alexa Fluor 750, and the label at the 3' end is selected from any one of TAMRA, Dabcyl, BHQ-1, BHQ-2, BHQ-3, MGB, or Eclipse.

[0020] Furthermore, the kit also includes one or more of the following components: PCR reaction solution, enzyme mixture, positive control, and negative control.

[0021] Furthermore, the kit also includes one or more of the following reagents: Tris-HCl buffer, MgCl2, dNTPs, KCl, NH4Cl, DNA polymerase, water, NaOH, Safrandine, DMSO, sucrose, and Chelex-100.

[0022] Furthermore, the test targets of the kit are whole blood, blood spots, oral swabs, saliva, amniotic fluid, or semen, etc.

[0023] For oral swabs and saliva, PCR amplification and detection can be performed directly without nucleic acid extraction. For other samples, nucleic acid extraction is required before testing.

[0024] Furthermore, the target of the test kit, oral swab samples, need to be placed in 0.5 mL-1 mL of oral swab lysis buffer after collection.

[0025] Furthermore, the kit is used in conjunction with digital PCR. The digital PCR program is set as follows: 50℃ pretreatment for 10 min, 95℃ pre-denaturation for 25 min, PCR cycle, 94℃ for 20 s, 56℃ for 40 s, 50 cycles.

[0026] As described above, the SMA detection kit based on digital PCR technology of the present invention has the following beneficial effects:

[0027] This invention distinguishes and accurately quantifies SMN1 and SMN2, simultaneously detecting the copy numbers of both SMN1 and SMN2 genes in the same reaction. It also adds detection of pathogenic point mutation sites in SMA and some other genes, improving diagnostic accuracy, simplifying procedures, and increasing timeliness. It can be applied to SMA carrier screening, clinical diagnosis, and disease subtyping, while also saving on testing costs. Attached Figure Description

[0028] Figure 1 The diagram shows the amplification direction of the primers and probes of this invention.

[0029] Figure 2A The image shows the reading results of the FAM channel.

[0030] Figure 2B The image shows the results of reading from the HEX channel.

[0031] Figure 2C The image shows the results of the ROX channel readings.

[0032] Figure 2D The image shows the reading results for the CY5 channel.

[0033] Figure 3A The image shows the reading results of the FAM channel.

[0034] Figure 3B The results are displayed as a scatter plot of HEX channel reads.

[0035] Figure 3C The results are displayed as a scatter plot of the ROX channel reads.

[0036] Figure 3D The results are displayed as a scatter plot of the CY5 channel readings.

[0037] Figure 4 This is one of the gene testing results of the child in Example 5.

[0038] Figure 5 This is the second image showing the genetic testing results of the child in Example 5. Detailed Implementation

[0039] Based on the pathogenic gene characteristics of SMA, existing gene diagnostic technologies mainly analyze whether there are deletions and the number of deletions in exon 7 and / or exon 8 of the SMN1 gene. However, exon 8 of the SMN1 gene does not participate in SMN protein expression, so detecting whether exon 8 of the SMN1 gene is deleted has little clinical significance. Furthermore, the primer-probe detection system designed in this invention, based on a digital PCR platform, can distinguish and accurately quantify SMN1 and SMN2 using only the MGB probe via c.840. It simultaneously detects the copy number of SMN1 and SMN2 genes in the same reaction, improving diagnostic accuracy, simplifying the operation process, and increasing timeliness. It can be applied to SMA carrier screening, clinical diagnosis, and disease subtyping, saving production costs.

[0040] To further optimize the technical solution, this application adds the detection of common pathogenic point mutation sites in SMA based on existing technologies. There are two main types of SMA mutation genotypes: 95% are caused by homozygous deletion of the SMN1 biallelic gene, i.e., the "0+0" genotype; 5% are caused by complex heterozygous mutations in SMN1, i.e., one allele is deleted and the other allele has a minor pathogenic variation, i.e., the "1d+1d" genotype, which is extremely rare, and currently only reported in cases of consanguineous marriage in Caucasians. This invention, based on simultaneously detecting the copy number of the SMN1 and SMN2 genes in the same reaction, adds a method for detecting common pathogenic point mutation sites in SMA, further improving diagnostic accuracy, simplifying the operation process, and increasing timeliness.

[0041] In existing clinical diagnoses of SMA, patients are tested and found to have an SMN1 copy number of 2, and it is necessary to exclude SMN1 microvariates and 5q-SMA. This necessitates further next-generation sequencing screening for other myasthenia gravis-related diseases. This invention, in the same reaction system, not only simultaneously detects the copy numbers of the SMN1 and SMN2 genes and common SMA pathogenic point mutation sites, but also detects other genes involved in non-5q spinal muscular atrophy, further improving diagnostic accuracy, simplifying the procedure, and increasing timeliness.

[0042] Furthermore, due to the high sensitivity and specificity of the probes and primers designed in this application, oral swabs or saliva samples from patients can be directly collected for testing without complex pretreatment processes. For example, the following procedure can be used: the prepared direct amplification lysis buffer is mixed with the sample at a 1:1 ratio, and the sample mixture is directly added to the PCR amplification reagent for digital PCR amplification. Lysis begins immediately after the sample is mixed with the direct amplification reagent. During pre-denaturation in the PCR instrument, the temperature is heated to 95°C, which ensures complete lysis of the sample. The nucleic acids released after sample lysis can then be used for PCR amplification, achieving direct amplification without sample extraction.

[0043] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or apparatus not specifically specified in the following embodiments are all conventional equipment or apparatus in the art. Furthermore, it should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated; it should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in the present invention does not exclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned two devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0044] Example 1: Construction of the reagent kit

[0045] I. Primers and Probes

[0046] 1. The upstream primer SEQ ID NO.1 for detecting the c.840 site of exon 7 of SMN1 (NG_008691.1:5001-33072) or SMN2 (NG_008728.1:5147-33073), and the downstream primer SEQ ID NO.2: TGAGCA CCT TCC TTC TTT;

[0047] 2. Probe SEQ ID NO.3 for detecting SMN1 exon 7: AGG GTT TCA GAC AAA;

[0048] 3. Probe SEQ ID NO.4 for detecting SMN2 exon 7: AGG GTT TTA GAC AAA;

[0049] 4. The upstream primer SEQ ID NO.5: TGTCAG CCT TAC ACA TCC, and the downstream primer SEQ ID NO.6: CAG CGC TAC AAT GGAGCA TCC, used for detecting the internal reference gene MRPS18C (NC_000004.12:83456058-83462298);

[0050] 5. Probe used to detect the internal reference gene SEQ ID NO.7: ACT CAC ACG GGT AAA GTC TCC.

[0051] 6. Primers and probes used to detect other gene mutations:

[0052] Table 1

[0053]

[0054] Each probe is labeled with a fluorescent generating group / fluorescence quenching group at both ends, and each probe is labeled with a different fluorescent group. For example, the fluorescent group labeled at the 5' end of the probe is FAM, FITC, Cy3, VIC, HEX, ROX, TET, NED, Cy5 or Cy5.5, and the label at the 3' end is MGB, BHQ-1, BHQ-2 or BHQ-3.

[0055] 7. Reagents required for PCR amplification

[0056] The buffer consists of Tris-HCl buffer (pH 8.3, 20 mmol / L), MgCl2, dNTPs, KCl, NH4Cl, DNA polymerase, and water.

[0057] Direct diffusion lysis buffer: NaOH 20 mmol / L, Safrandine 0.5 wt.%, DMSO 5 wt.%, sucrose 8 wt.%, Chelex-100 0.9 wt.%.

[0058] II. Target of Testing

[0059] Whole blood, blood spots, oral swabs, saliva, amniotic fluid, semen, etc.

[0060] For oral swabs and saliva, PCR amplification and detection can be performed directly without extracting nucleic acid.

[0061] For other samples, nucleic acid needs to be extracted and then tested.

[0062] III. Digital PCR Setup Procedure:

[0063] Pre-treat at 50℃ for 10 min, pre-denature at 95℃ for 25 min, then enter PCR cycle, 94℃ for 20 s, 56℃ for 40 s, for 50 cycles.

[0064] IV. Interpretation of Results

[0065] 1. SMN1 gene interpretation: (FAM signal is involved in the interpretation)

[0066] (1) Sites with FAM signal and a relative copy number (internal reference gene copy number is defined as 2) of 2 are considered wild-type SMN1; (normal)

[0067] (2) Points without FAM signal are patients with homozygous deletion of SMA; (homozygous deletion) accounts for 95%, and 5% are compound heterozygous mutations;

[0068] (3) Points with FAM signal and a relative copy number (internal reference gene copy number is defined as 2) of 1 are SMA carriers with SMN1 gene deletion;

[0069] 2. SMN2 gene interpretation: (ROX and HEX signals are involved in the interpretation)

[0070] (1) HEX has a signal, which is SMN2 wild; used for copy number quantification;

[0071] (2) Points with only ROX signal and no HEX signal are where SMN2 abruptly changes to SMN1;

[0072] (3) Points with only HEX signal and no ROX signal are where SMN1 changes to SMN2.

[0073] When the internal reference gene is a 2-copy gene, the absolute copy number of the test gene is determined by the following numerical PCR ratio between the test gene and the internal reference gene. A positive signal from a detection probe for common SMA pathogenic point mutations indicates the presence of SMA pathogenic point mutations in the sample. A positive signal from a detection probe for other genes involved in non-5q spinal muscular atrophy indicates the presence of other gene mutations involved in non-5q spinal muscular atrophy in the sample.

[0074] Table 2

[0075]

[0076] Example 2: Reagent Specificity and Sensitivity

[0077] Specific experiments

[0078] 1. Synthesize double-stranded DNA containing the human SMN1 gene as the core sequence: Clone to the PMD9-T vector via TA cloning, then clone, sequence verification, and determine the plasmid solution concentration to obtain a plasmid containing the SMN1 gene (SN1) as a nucleic acid detection standard; similarly, prepare plasmids containing the SMN2 gene (SN2), E1 c.22dupA (M1), E3 c.400G>A (M2), E3 c.463_464delAA (M3), E5 c.683T>A (M4), E5 c.689C>T (M5), I6 c.835-5T>C (M6), and E7... The plasmids c.863G>T (M7), G1 (containing the ZPR1 gene), G2 (containing the NAIP gene), G3 (containing the PLS3 gene), and G4 (containing the NCALD gene) were prepared. Physiological saline was used as the NTC, and equal amounts of the constructed plasmids SN1–G4 were mixed as the PTC.

[0079] 2. Reagent preparation

[0080] Prepare dPCR reaction solution for N+3* reactions according to the table below (1 reaction / tube, 30 μL / reaction).

[0081] Table 3

[0082]

[0083] *N represents the number of samples to be tested; 3 represents one positive control, one negative control, and an extra sample is taken to avoid volume loss due to sample addition error.

[0084] 3. Chip Sample Introduction

[0085] Prepare the oil phase for the reaction according to the table below:

[0086] Table 4

[0087]

[0088] Add oil phase A and oil phase B to oil tank A1 and oil tank B1 of the oil tank plate respectively according to the table above; then install the oil tank plate in the oil tank plate position of the fully automatic sample processing system and fix it.

[0089] 4. Chip Amplification

[0090] After sample injection, remove the chip; then place the chip in the chip holder of the PCR amplification instrument for thermal cycling amplification. The recommended reaction procedure is as follows:

[0091] Table 5

[0092]

[0093] 5. Chip Reading and Analysis

[0094] After the thermal cycling reaction was completed, the chip was transferred to a biochip reader, and a four-channel reading plan (FAM, HEX, ROX, and CY5) was established, with the experimental type set to "DQ". The results are as follows: Figure 2A-2D .

[0095] 6. Results Analysis

[0096] Table 6

[0097]

[0098] The above analysis shows that all prepared standards can be detected stably and have good specificity.

[0099] Linearity Experiment

[0100] 1. Take a quantitative amount of PTC plasmid and dilute the sample 5 times to obtain quantitative values ​​of 5 ng / uL, 25 ng / uL, 50 ng / uL, and 75 ng / uL, and label them as 5 ng, 25 ng, 50 ng, and 75 ng for experiments.

[0101] 2. Detection Method

[0102] Conduct the experiment according to the above operating steps.

[0103] 3. Results Analysis

[0104] The results are as follows Figures 3A-3D It can be seen that the test results and the theoretical values ​​satisfy a linear relationship R2≧0.980, indicating excellent linearity.

[0105] Sensitivity Experiment

[0106] 1. Take a quantitative amount of PTC plasmid, dilute it to 500 copies / µL, label it as S0, and conduct experiments.

[0107] 2. Detection Method

[0108] Repeat the above procedure 10 times for S0.

[0109] 3. Results Analysis

[0110] Table 7

[0111]

[0112] The above analysis shows that at a concentration of 500 copies / uL, the positive detection rate reaches 90%, indicating high sensitivity.

[0113] Example 3: Detection of oral swabs (direct amplification), oral swabs with nucleic acid extraction, and blood samples.

[0114] 1. Sample Source

[0115] Oral swab samples and anticoagulated peripheral blood samples were collected from healthy individuals (WMY, HXJ) at the same time.

[0116] 2. Sample processing

[0117] a. Direct amplification of oral swabs: Add the oral swab preservation solution to the prepared direct amplification lysis buffer and mix with the oral swab sample at a ratio of 1:1.

[0118] b. Oral mucosal sample: Nucleic acid extraction was performed using 200 μL of human oral mucosal sample. A commercially available extraction kit is recommended: Salivary Genome Extraction Kit (Beaver). Please refer to the instructions for the appropriate extraction kit for specific extraction steps.

[0119] c. Human anticoagulated peripheral blood samples (WMY, HXJ): Nucleic acid extraction was performed using 200 μL of human anticoagulated peripheral blood sample. A commercially available extraction kit is recommended: Blood Genome Extraction Kit (Beaver). Please refer to the instructions for the appropriate extraction kit for specific extraction steps.

[0120] 3. The experimental steps were the same as in Example 4, and the results are as follows:

[0121] Table 8

[0122]

[0123] The results show that the present invention can accurately identify the copy number of SMN1 and SMN2 genes, as well as micromutations and other gene mutations, in both direct amplification samples of oral swabs, samples of oral swabs extracted with nucleic acid, and peripheral blood samples.

[0124] Example 4: Multi-sample analysis of actual clinical samples

[0125] 1. Sample

[0126] Human oral mucosa samples: Nucleic acid extraction was performed using 200 μL of human oral mucosa sample. Negative controls need to be extracted along with the clinical samples. A commercially available extraction kit is recommended: Salivary Genome Extraction Kit (Beaver). Please refer to the corresponding extraction kit's instructions for specific extraction steps.

[0127] The nucleic acid extraction solution obtained after extraction was detected by ultraviolet spectrophotometer. The OD260 / OD280 should be between 1.6 and 2.0, and the nucleic acid concentration should be around 5 to 20 ng / μL.

[0128] 2. Reagent preparation

[0129] Take 5×Hi-dPCR Buffer, Hi-dPCR enzyme, and SMA detection system from the kit, thaw and mix them at room temperature, then centrifuge briefly and set aside. Prepare N+3* dPCR reaction solutions in eight-tube arrays according to the table below (1 reaction / tube, 30μL / reaction).

[0130] Table 9

[0131]

[0132] *N represents the number of samples to be tested; 3 represents one positive control, one negative control, and an extra sample is taken to avoid volume loss due to sample addition error.

[0133] 3. Adding samples

[0134] Take 5 μL of the nucleic acid extraction solution of the sample and negative control from step 1, and add 5 μL of the positive control to each dPCR reaction solution (the sample loading amount should be 25~100 ng). Mix slowly by pipetting for 30 seconds. Then seal and centrifuge for 3 seconds for later use.

[0135] Finally, remove the eight-tube cap, install it in the 96-well plate position of the fully automated sample processing system, and secure it.

[0136] 4. Chip Sample Introduction

[0137] Prepare the oil phase for the reaction according to the table below: Table 10

[0138]

[0139] Add oil phase A and oil phase B to oil tank A1 and oil tank B1 of the oil tank plate respectively according to the table above; then install the oil tank plate in the oil tank plate position of the fully automatic sample processing system and fix it.

[0140] 5. Chip Amplification

[0141] After the chip injection is complete, remove the chip; then place the chip in the chip holder of the PCR amplification instrument for thermal cycling amplification. The reaction procedure is as follows:

[0142] Table 11

[0143]

[0144] 6. Chip Reading and Analysis

[0145] After the thermal cycling reaction, the chip was transferred to a biochip reader, and a five-channel reading plan (FAM, HEX, ROX, CY5, and CY5.5) was established, with the "DQ" experimental type set. The formulas for calculating the relative copy number of SMN1 and SMN2 are as follows:

[0146]

[0147]

[0148] Then, the reading and analysis were performed according to the instructions of the biochip reader; and the sample template concentration and mutation rate were calculated.

[0149] 7. Data Analysis

[0150] Table 12

[0151] The results showed that the detection rate and specificity of this invention in SMA patients were 100%. Among them, the SMA1157 sample showed a heterozygous deletion of the SMN1 gene but with a small mutation, therefore it was judged as "0+1". d The SMA2613 sample was identified as a heterozygous deletion type in SMN1 gene testing, with no micromutations or other modified gene mutations. Based on this, it was determined to be an SMA carrier.

[0152] The SMA5113 sample tested homozygous wild-type SMN1 gene and showed no micromutations, but other modified gene mutations were present, leading to a diagnosis of non-5q-SMA. Other samples tested homozygous deletion type SMN1, indicating SMA. SMN2 copy number tests also confirmed clinical presentation.

[0153] Example 5: Specific Case of Clinical Application of the Reagent Kit

[0154] A 3-year-old male infant, a patient with SMA1157, presented with swallowing and sucking dysfunction and muscle weakness after birth, and sought medical help from various sources. However, a definitive diagnosis could not be obtained. Later, at the First Affiliated Hospital of Zhengzhou University, the present invention was used for testing, revealing a tiny mutation, which was verified by Sanger sequencing as E6 c.835-5T>G. This confirmed that the infant suffered from the internationally rare SMN1 mutation "0+1d" genotype. Simultaneously, the First Affiliated Hospital of Zhengzhou University conducted clinical evaluations and examinations, including pathological, neurological, and motor function tests, providing strong clinical diagnostic support.

[0155] b. SMA2613, female, 7 years old. She presented with dysphagia, choking while feeding, and muscle weakness from birth, and sought medical help from various sources. In March 2022, the child's mother, presenting with the chief complaint of "17 weeks of pregnancy, history of having a child with SMA," sought genetic counseling and prenatal diagnosis at the First Affiliated Hospital of Zhengzhou University. Previous MLPA results from multiple hospitals indicated that the child had SMA caused by homozygous deletion of the SMN1 gene E7 and had 2 copies of the SMN2 gene (…). Figure 4 However, the child was still alive at the age of 7, which did not conform to conventional understanding. Zhengzhou University First Affiliated Hospital used the present invention to test and found that the child's SMN1 gene E7 was homozygous deletion, indicating that he was an SMA carrier, which explained the contradiction between the clinical manifestations and the gene test.

[0156] Sequencing results of the SMN1 gene E7 in the child from Zhengda First Affiliated Hospital showed that the c.840 position was C, but there was a mutation in the intron region (the location of the MLPA probe), which prevented the MLPA probe from binding, resulting in a false positive result. Figure 5 ).

[0157] The child was ultimately diagnosed with rod body myopathy caused by a complex heterozygous variant of the NEB gene, rather than SMA, thus identifying a potential pitfall in genetic diagnosis that could lead to a misdiagnosis as SMA. Furthermore, by following the prenatal diagnostic approach for rod body myopathy rather than SMA, the family obtained accurate fetal genetic testing results, enabling precise prevention and avoiding the birth of an affected fetus.

[0158] c.SMA5113, female, 10 months old. She cannot roll over, has difficulty lifting her head while prone, dislikes lying on her stomach, cannot crawl, cannot sit for long periods, has weakness in her limbs, her body is limp, her legs cannot straighten when standing, and has high muscle tone in her lower limbs. She actively grasps objects with both hands, but her arms cannot support her upper body to lift her head. When pulled up to sit, she can lift her head, but her feet remain on the ground. The child's responsiveness, IQ, and language function are normal. She sought medical help from various sources, but without a definitive diagnosis. At the First Affiliated Hospital of Zhengzhou University, testing using this invention revealed mutations in other modified genes. NGS sequencing confirmed a deletion in exon 5 of the NAIP gene, confirming that the child has non-5q-SMA. Simultaneously, the First Affiliated Hospital of Zhengzhou University conducted clinical evaluations and examinations, including pathological, neurological, and motor function assessments, providing strong clinical support for the diagnosis.

[0159] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A kit for detecting SMN1 and SMN2 genes using digital PCR technology, characterized in that: The kit contains the following probes and primers: a. An upstream primer for detecting the 7th exon of SMN1 or SMN2, the nucleotide sequence of which is shown in SEQ ID NO.1, and a downstream primer, the nucleotide sequence of which is shown in SEQ ID NO.2; b. A probe for detecting exon 7 of SMN1, the nucleotide sequence of which is shown in SEQ ID NO.3; c. A probe for detecting exon 7 of SMN2, the nucleotide sequence of which is shown in SEQ ID NO.4; and d. The following probes and primers are used to detect target genes: Each of the probes described above has a pair of distinct fluorescent reporter groups and fluorescent quencher groups attached to both ends.

2. The reagent kit according to claim 1, characterized in that: The kit also includes primers and probes for detecting the internal reference gene.

3. The reagent kit according to claim 2, characterized in that: The internal reference gene is MRPS18C. The upstream primer for detecting the internal reference gene MRPS18C has the nucleotide sequence shown in SEQ ID NO.5; the downstream primer has the nucleotide sequence shown in SEQ ID NO.6; and the probe nucleotide sequence is shown in SEQ ID NO.

7.

4. The kit according to claim 1 or 3, characterized in that: The fluorescent group labeled at the 5' end of the probe is selected from any one of FAM, HEX, TET, ROX, CY3, CY5, CY5.5, VIC, JOE, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 647, or Alexa Fluor 750, and the label at the 3' end is any one of TAMRA, Dabcyl, BHQ-1, BHQ-2, BHQ-3, MGB, or Eclipse.

5. The reagent kit according to claim 1, characterized in that: The kit also includes one or more of the following components: PCR reaction solution, enzyme mixture, positive control or negative control.

6. The reagent kit according to claim 1, characterized in that: The kit also includes one or more of the following reagents: Tris-HCl buffer, MgCl2, dNTPs, KCl, NH4Cl, DNA polymerase, water, NaOH, Safran, DMSO, sucrose, and Chelex-100.

7. The kit according to claim 1, characterized in that: The kit can be used to detect whole blood, blood spots, oral swabs, saliva, amniotic fluid, or semen.

8. The reagent kit according to claim 1, characterized in that: For the test subjects of the kit, oral swab samples need to be placed in 0.5 mL-1 mL of oral swab lysis buffer after collection.

9. The reagent kit according to claim 1, characterized in that: All primers and probes described in the kit are contained in a single reaction system.

10. The kit according to claim 1, characterized in that: The kit is used in conjunction with digital PCR. The digital PCR program is set as follows: 50℃ pretreatment for 10 min, 95℃ pre-denaturation for 25 min, PCR cycle, 94℃ for 20 s, 56℃ for 40 s, 50 cycles.

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