Use of SCN1A in preparation of kit for detecting neurodevelopmental disorder disease

Abstract

The present application relates to the field of detection reagents for neurodevelopmental disorders, and in particular to the application of SCN1A in the preparation of a kit for detecting neurodevelopmental disorders, especially the application of specific multiple mutation sites of the gene. These mutation sites include single nucleotide deletion mutations of chr2:165984651, chr2:165984876 and chr2:165984932, which are associated with attention deficit hyperactivity disorder. The kit provided by the present application combines targeted cleavage and fluorescent PCR amplification technology to improve the detection sensitivity of multiple mutation sites of the SCN1A gene. The kit provided by the present application covers multiple SCN1A gene mutation sites, enhances the comprehensiveness of detection, and recognizes complex mutation individuals through threshold effect, thereby improving the sensitivity and specificity of detection.

Description

TECHNICAL FIELD

[0001] The application relates to the field of detection reagents for neurodevelopmental disorders, and in particular to application of SCN1A in preparation of a kit for detecting neurodevelopmental disorders. BACKGROUND

[0002] A neurodevelopmental disorder is a disease that causes persistent functional disorders in cognition, behavior, language, movement, etc. due to abnormal or impaired development of the nervous system during the fetal period to the adolescence. Attention deficit hyperactivity disorder (ADHD), also known as childhood hyperactivity, is one of the most common neurodevelopmental disorders, and is mainly manifested in inattention, hyperactivity and impulsivity, which are not commensurate with the age and development level, and are often accompanied by learning difficulties, conduct disorders and maladjustment. As a neurodevelopmental disorder, the main characteristics of ADHD are inattention, hyperactivity and impulsivity, which usually occur in childhood and may continue into adulthood. According to the International Classification of Diseases (ICD-11) and the American Diagnostic and Statistical Manual of Mental Disorders (DSM-5), ADHD is clearly classified as a neurodevelopmental disorder.

[0003] The voltage-gated sodium ion channel alpha subunit type 1 (SCN1A) gene is located at 2q24.3 of the long arm of chromosome 2, has a full length of about 70 kb, contains 26 exons, encodes Nav1.1 protein, and is mainly expressed in the central nervous system, and plays an important role in regulating the excitability of neurons and the generation of action potentials. Research has found that loss-of-function mutations of the SCN1A gene are related to various nervous system diseases, including ADHD, epilepsy, febrile convulsions, etc. At present, the detection methods of the SCN1A gene in the clinic mainly include Sanger sequencing, second-generation sequencing, high-resolution melting curve method, fluorescence quantitative PCR, etc. These methods have advantages and disadvantages, such as low sensitivity and low throughput of Sanger sequencing, high cost and complex data analysis of second-generation sequencing, high requirement for instruments and strong subjectivity of result interpretation of the high-resolution melting curve method, poor specificity and easy false positives of fluorescence quantitative PCR, etc. Therefore, it has important clinical significance to develop a detection kit and method for SCN1A gene multiple mutation sites with high specificity, high sensitivity, low cost and simple operation. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides application of SCN1A in preparation of a kit for detecting neurodevelopmental disorders.

[0005] In a first aspect, the present application provides a kit for detecting a neurodevelopmental disorder, comprising a CRISPR / Cas9 cleavage system and a fluorescent PCR amplification system. The CRISPR / Cas9 cleavage system comprises sgRNA-1 as shown in SEQ ID NO. 2, sgRNA-2 as shown in SEQ ID NO. 3, sgRNA-3 as shown in SEQ ID NO. 4, and Cas9 protein. The fluorescent PCR amplification system comprises DNA molecules as shown in SEQ ID NO. 5-13. The CRISPR / Cas9 cleavage system targets and cleaves genomic DNA to obtain a target sequence comprising SCN1A gene multiple mutation sites, the fluorescent PCR amplification system amplifies the target sequence, the SCN1A gene multiple mutation sites are single nucleotide deletion mutations at chr2:165984651, chr2:165984876, and chr2:165984932, and the neurodevelopmental disorder is specifically attention deficit hyperactivity disorder.

[0006] In some embodiments, the CRISPR / Cas9 cleavage system specifically comprises Cas9 protein at a final concentration of 50-200 nM, sgRNA-1 at a final concentration of 50-300 nM, sgRNA-2 at a final concentration of 50-300 nM, sgRNA-3 at a final concentration of 50-300 nM, 50-200 ng of genomic DNA, 10×NEBuffer3.1, BSA at a final concentration of 0.05-0.15 mg / mL, RNase Inhibitor at a final concentration of 0.1-1 U / μL, and double distilled water.

[0007] In some embodiments, the CRISPR / Cas9 cleavage system specifically comprises Cas9 protein at a final concentration of 100 nM, sgRNA-1 at a final concentration of 200 nM, sgRNA-2 at a final concentration of 200 nM, sgRNA-3 at a final concentration of 200 nM, 100 ng of genomic DNA, 10×NEBuffer3.1, BSA at a final concentration of 0.1 mg / mL, RNase Inhibitor at a final concentration of 0.5 U / μL, and double distilled water.

[0008] In some embodiments, the DNA as shown in SEQ ID NO. 5 and the DNA as shown in SEQ ID NO. 6 are a primer pair, and the DNA as shown in SEQ ID NO. 7 is a TaqMan probe.

[0009] In some embodiments, the DNA as shown in SEQ ID NO. 8 and the DNA as shown in SEQ ID NO. 9 are a primer pair, and the DNA as shown in SEQ ID NO. 10 is a TaqMan probe.

[0010] In some embodiments, the DNA as shown in SEQ ID NO. 11 and the DNA as shown in SEQ ID NO. 12 are a primer pair, and the DNA as shown in SEQ ID NO. 13 is a TaqMan probe.

[0011] In some embodiments, the fluorescent PCR amplification system comprises, in 100 μL, 5-10 ng of the cleavage product DNA, 12.5 μL of 2x TaqMan Master Mix, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 5, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 6, 0.2 μM of the DNA molecule as shown in SEQ ID NO. 7, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 8, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 9, 0.2 μM of the DNA molecule as shown in SEQ ID NO. 10, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 11, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 12, 0.2 μM of the DNA molecule as shown in SEQ ID NO. 13, and double distilled water.

[0012] In some embodiments, a triphosphate (PPP) label is added to the 5' end of the sgRNA-1 as shown in SEQ ID NO. 2, a triphosphate (PPP) label is added to the 5' end of the sgRNA-2 as shown in SEQ ID NO. 3, and a triphosphate (PPP) label is added to the 5' end of the sgRNA-3 as shown in SEQ ID NO. 4.

[0013] In the second aspect, the application provides use of SCN1A in the preparation of the kit of the first aspect. Specifically, use of the SCN1A gene multiple mutation sites in the preparation of a kit for detecting a neurodevelopmental disorder is provided. The SCN1A gene multiple mutation sites are single nucleotide deletion mutations at chr2:165984651, chr2:165984876 and chr2:165984932, and the neurodevelopmental disorder is specifically attention deficit hyperactivity disorder.

[0014] Compared with the prior art, the application has the following beneficial effects:

[0015] The application successfully guides the Cas9 enzyme to precisely cut the target genomic DNA by using the specifically designed gRNA, thereby significantly improving the specificity of detection. Experimental data show that the false positive rate of the method of the application is greatly reduced compared with the traditional fluorescent PCR amplification method.

[0016] The detection method of the present application combines targeted cleavage with fluorescence PCR amplification technology, significantly improving the sensitivity of detection. This advantage enables the present application to still provide accurate detection results when facing trace amounts of target sequences.

[0017] The present application obtains a complete fragment containing all three mutation sites through one CRISPR cleavage, simplifying the detection process and avoiding the complexity of multi-fragment detection. In addition, the present application only requires a small amount of DNA sample, which is particularly suitable for the detection of precious samples such as circulating free DNA (cfDNA), greatly reducing the demand for sample size.

[0018] The multiplex qPCR technology used in the present application detects three sites in a single tube, effectively reducing operation errors and reagent consumption. In addition, the use of fluorescent probes further improves the specificity of detection, avoiding the interference of non-specific amplification and ensuring the accuracy of the detection results.

[0019] The combined detection method of the present application can simultaneously evaluate multiple pathogenic mechanisms through complementary coverage, improving the comprehensiveness of detection. The present application utilizes the threshold effect, so that even if a single site mutation is not enough to trigger clinical symptoms, the combined detection can identify individuals with compound mutations, thereby improving the sensitivity and specificity of detection.

[0020] Through combined detection, the present application significantly improves the detection efficiency, with a sensitivity improvement of 20%-25% and a misdiagnosis rate reduction from 27%-32% to 7%. At the same time, the specificity is improved from 84%-87% to 95%, effectively eliminating false positives of a single site and improving the accuracy of detection.

[0021] The combined detection kit and method of the present application can accurately identify different disease subtypes. For example, in patients with attention deficit hyperactivity disorder (ADHD), the positive rate of combined detection is significantly higher than that of single site detection, thereby more comprehensively covering the genetic heterogeneity of ADHD. In terms of early detection, the present application also shows significant advantages. In patients with mild ADHD, the positive rate of combined detection is significantly higher than that of single site detection. Follow-up studies of high-risk children also show that the proportion of ADHD diagnosed within 2 years in individuals with positive combined detection is much higher than that in individuals with single site detection, indicating that the present application has important application value in early detection. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The cleavage products of genomic DNA for experimental group 1 (lane 1), experimental group 2 (lane 2), experimental group 3 (lane 3), and experimental group 4 (lane 4) are electrophoresed.

[0023] Figure 2 The columnar results of editing efficiency for experimental group 1, experimental group 2, experimental group 3, and experimental group 4 are shown.

[0024] Figure 3 The ROC curves of site 1 alone detection (A), site 2 alone detection (B), site 3 alone detection (C) and three-site combined detection (D) are shown in Figure 1. DETAILED DESCRIPTION

[0025] In order to make the objects, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application. The reagents not specifically described in the present application are conventional reagents and can be obtained from commercial channels; the methods not specifically described are conventional experimental methods and can be known from prior art.

[0026] The present application designs three sgRNAs (single guide RNA) according to the location of the SCN1A gene in the chromosome and taking the sequence shown as SEQ ID NO. 1 as the target sequence. In view of the specific DNA sequence recognition in the CRISPR / Cas9 system and the cutting reaction characteristics of the Cas9 / sgDNA complex, the present application designs a CRISPR / Cas9 detection system and a method for detecting SCN1A gene mutations based on the CRISPR / Cas9 detection system, and combines with the fluorescence quantitative PCR (qPCR) to detect the single nucleotide deletion mutation at chr2:165984651 (GRCh38.p14), chr2:165984876 (GRCh38.p14) and chr2:165984932 (GRCh38.p14) in the SCN1A gene, and then determines the detection of attention deficit hyperactivity disorder in neurodevelopmental disorders according to the genotype of the detected deletion mutation site.

[0027] I. CRISPR / Cas9 cutting system

[0028] The present application provides a CRISPR / Cas9 cutting system, which comprises sgRNA-1 shown as SEQ ID NO. 2, sgRNA-2 shown as SEQ ID NO. 3, sgRNA-3 shown as SEQ ID NO. 4 and Cas9 protein. The present application uses the cutting system to perform targeted cutting on genomic DNA to obtain the target fragment at chr2:165984651, chr2:165984876 and chr2:165984932 in the SCN1A gene, thereby improving the efficient acquisition of single nucleotide deletion mutation in the target fragment, and further improving the detection sensitivity of the mutation site in the target fragment and improving the detection efficiency.

[0029] The CRISPR / Cas9 cutting system provided by the application is characterized in that sgRNA-1 targets a site upstream of chr2:165984651 (SEQ ID NO. 2: 5'-AUUAUGCAUGUGUAUAUUUU-3'), sgRNA-2 targets a site downstream of chr2:165984932 (SEQ ID NO. 3: 5'-AGGGCUCUAUACUCACUACCU-3'), and sgRNA-3 targets a site upstream of chr2:165984876 (SEQ ID NO. 4: 5'-CUUUCCCAGUAACAUUAUGAG-3'). The sgRNA-1 and sgRNA-2 jointly act, the sgRNA-3 assists in cutting, the complete 524bp target fragment (as shown in SEQ ID NO. 1) is released, the cutting efficiency is enhanced, and the detection sensitivity is improved.

[0030] Specifically, the application provides a CRISPR / Cas9 cutting system, which comprises Cas9 protein with a final concentration of 50-200nM, sgRNA-1 with a final concentration of 50-300nM, sgRNA-2 with a final concentration of 50-300nM, sgRNA-3 with a final concentration of 50-300nM, 50-200ng of genomic DNA, 10x NEBuffer 3.1, BSA with a final concentration of 0.05-0.15mg / mL, Rnase Inhibitor with a final concentration of 0.1-1U / μL, and double-distilled water.

[0031] The genomic DNA extracted by a conventional method is incubated with the cutting system to obtain an incubated product, the cutting product is detected by electrophoresis, and the cutting product is amplified by fluorescent quantitative PCR, and whether the gene to be detected is mutated is determined according to the fluorescent quantitative PCR result.

[0032] In some embodiments, the incubation temperature of the cutting system is preferably 35-38℃, and the incubation time is preferably 55-65min, which is beneficial to improve the Cas9 cutting efficiency, and further improve the accuracy and sensitivity of the detection method.

[0033] In order to improve the in vitro cutting efficiency and stability, the application further provides sgRNA-1 with a 5' end adding triphosphate (PPP) label, sgRNA-2 with a 5' end adding triphosphate (PPP) label, and sgRNA-3 with a 5' end adding triphosphate (PPP) label, which enhances the binding affinity with Cas9 protein. The method for adding triphosphate (PPP) label to the 5' end is referred to "Protocol for 5' End Labeling RNA|Thermo Fisher Scientific-US" of Thermo Fisher Scientific.

[0034] The three sgRNAs provided by the application cooperatively cut the genomic DNA to release a fragment of about 524 bp containing three mutation sites, so as to facilitate the detection of the genotypes of the three sites by designing three pairs of primers and fluorescent probes for quantitative PCR using the cutting product as a template. The wild-type probe binds to the complete sequence, and the deletion mutation causes the probe signal to weaken or disappear, thereby improving the detection efficiency.

[0035] Evaluation of cutting efficiency:

[0036] Experimental group 1: 100 nM of Cas9 protein (Yunzhou Biotechnology) was added to a final concentration, 200 nM of sgRNA-1 was added to a final concentration, 200 nM of sgRNA-2 was added to a final concentration, 200 nM of sgRNA-3 was added to a final concentration, 100 ng of genomic DNA was added, 2.5 μL of 10×NEBuffer3.1 was added, 0.1 mg / mL of BSA was added to a final concentration, 0.5 U / μL of RNase Inhibitor was added to a final concentration, and the rest was ddH2O to a total volume of 25 μL. Incubation was performed at 37°C for 60 min, and after inactivation at 80°C for 10 min, 1.5% agarose gel electrophoresis was performed. The stained gel was imaged using a gel imaging system (such as a GelDoc system) or a fluorescence imager, and the generation of a 524 bp band was observed. The editing efficiency was calculated by analyzing the intensity of the 524 bp electrophoretic band. The editing efficiency can be calculated by the formula: editing efficiency = I

[0037] ; wherein, I 切割 is the intensity of the about 524 bp electrophoretic band, and I 总 is the sum of the intensities of all bands.

[0038] Experimental group 2: 100 nM of Cas9 protein was added to a final concentration, 200 nM of sgRNA-1 was added to a final concentration, 200 nM of sgRNA-2 was added to a final concentration, 100 ng of genomic DNA was added, 2.5 μL of 10×NEBuffer3.1 was added, 0.1 mg / mL of BSA was added to a final concentration, 0.5 U / μL of RNase Inhibitor was added to a final concentration, and the rest was ddH2O to a total volume of 25 μL. Incubation was performed at 37°C for 60 min, and after inactivation at 80°C for 10 min, the detection steps were the same as those of experimental group 1.

[0039] Experimental Group 3: Cas9 protein (final concentration 100 nM), sgRNA-1 (final concentration 200 nM), genomic DNA (100 ng), 1.5 μL 10×NEBuffer 3.1, BSA (final concentration 0.1 mg / mL), RNase Inhibitor (final concentration 0.5 U / μL), and ddH2O were added to bring the total volume to 25 μL. The mixture was incubated at 37℃ for 60 min, then inactivated at 80℃ for 10 min. The detection procedure was the same as in Experimental Group 1.

[0040] Experimental Group 4: Cas9 protein (final concentration 100 nM), sgRNA-2 (final concentration 200 nM), genomic DNA (100 ng), 1.5 μL 10×NEBuffer 3.1, BSA (final concentration 0.1 mg / mL), RNase Inhibitor (final concentration 0.5 U / μL), and ddH2O were added to bring the total volume to 25 μL. The mixture was incubated at 37°C for 60 min, then inactivated at 80°C for 10 min. The detection procedure was the same as in Experimental Group 1.

[0041] like Figure 1 As shown, experimental groups 1 and 2 both showed a target band of approximately 524 bp, while experimental groups 3 and 4 did not show a target band. However, the target band in experimental group 1 was brighter, indicating that experimental groups 1 and 2 were able to cut genomic DNA, while experimental groups 3 and 4 were not.

[0042] like Figure 2 As shown, the editing efficiency of experimental group 1 is higher than that of experimental group 2.

[0043] II. Quantitative Real-Time PCR Detection

[0044] 1. Primer and probe sequence design

[0045] Specific primers and probes were designed for the three mutation sites in the cleaved fragment, as shown in Table 1. In Table 1, the primer pairs shown in SEQ ID NO.5 and SEQ ID NO.6 and the wild-row probe shown in SEQ ID NO.7 were used to amplify the target sequence containing the delG-165984651 site; the primer pairs shown in SEQ ID NO.8 and SEQ ID NO.9 and the wild-row probe shown in SEQ ID NO.10 were used to amplify the target sequence containing the delC-165984876 site; and the primer pairs shown in SEQ ID NO.11 and SEQ ID NO.12 and the wild-row probe shown in SEQ ID NO.13 were used to amplify the target sequence containing the delG-165984932 site.

[0046] Table 1 Primers and probes

[0047]

[0048] 2. Detection principle

[0049] In wild-type samples, the primer pair of the fluorescent quantitative PCR can amplify the cleavage product obtained by the CRISPR / Cas9 cleavage system, and the three different fluorescently labeled probes can effectively bind to the PCR amplification product, so that the fluorescence signals of the three channels of FAM, VIC and ROX can be normally displayed, and maintained at an expected level.

[0050] For samples containing deletion mutations, the presence of mutation sites will interfere with the complete hybridization of probes to wild-type target sequences. Due to the inability of probes to correctly bind to the mutated sequence, this incomplete hybridization will cause the signal intensity of the fluorescence channel corresponding to the mutation site to weaken, and even possibly completely disappear.

[0051] 3. Multiplex qPCR

[0052] Table 2 Reaction system (100 μL)

[0053] Component Concentration Cutting product DNA 5-10 ng 2x TaqMan Master Mix 12.5 μL Primer mix (three pairs) 0.4 μM each Probe mix (FAM / VIC / ROX) 0.2 μM each RNase-free water Supplemented to 100 μL

[0054] The fluorescent PCR detection method is as follows: first prepare 100 μL of reaction system, which contains 5-10 ng of cleavage product DNA as template, 2x TaqMan Master Mix (containing Taq enzyme and dNTPs, SR2110, Solarbio), and three pairs of primer mixtures (0.4 μM each) for amplifying three target regions. In addition, 0.2 μM of probe mixture (FAM / VIC / ROX) is also added for detecting wild-type sequence. Finally, use RNase-free water to make up to 100 μL. In the thermal cycling conditions, first pre-denature at 95℃ for 10 minutes, then perform multiple cycles, each cycle including 95℃ denaturation for 15 seconds and 60℃ annealing + extension for 1 minute, during which the fluorescence signal is collected.

[0055] 4. Genotype interpretation criteria

[0056] Table 3

[0057] Genotype FAM signal (delG-165984651) VIC signal (delC-165984876) ROX signal (delG-165984932) Wild type homozygote Ct < 30, high fluorescence intensity Ct < 30, high fluorescence intensity Ct < 30, high fluorescence intensity Deletion homozygote No Ct value or Ct > 35 No Ct value or Ct > 35 No Ct value or Ct > 35 Heterozygote 30 ≤ Ct ≤ 35, medium intensity 30 ≤ Ct ≤ 35, medium intensity 30 ≤ Ct ≤ 35, medium intensity

[0058] III. Actual sample detection

[0059] 1. Detection method

[0060] Samples: Select the known genotype of genomic DNA samples, including normal samples without chr2: 165984651, chr2: 165984876 and chr2: 165984932 three mutation sites, homozygous deletion mutant sample 1 containing chr2: 165984651 mutation site, homozygous deletion mutant sample 2 containing chr2: 165984876 mutation site, homozygous deletion mutant sample 3 containing chr2: 165984932 mutation site, homozygous deletion mutant sample 4 containing chr2: 165984651, chr2: 165984876 and chr2: 165984932 three mutation sites.

[0061] Experimental group:

[0062] Mix 100 nM Cas9 protein, 200 nM sgRNA-1, 200 nM sgRNA-2, 200 nM sgRNA-3 and 100 ng genomic DNA with a final concentration of 0.1 mg / mL BSA, 0.5 U / μL RNase Inhibitor and double distilled water, total volume 500 μL, 37℃ incubation for 60 min, 1.5% agarose gel electrophoresis, recovery of the target band, which is the cleavage product. Prepare the reaction system as shown in Table 2, under thermal cycling conditions, first denature at 95℃ for 10 minutes, then 40 cycles, each cycle includes 95℃ denaturation for 15 seconds and 60℃ annealing + extension for 1 minute, during which the fluorescence signal is collected. According to the fluorescence amplification curve, the Ct values of FAM signal, VIC signal and ROX signal are obtained respectively, and whether each position exists mutation site is determined according to the judgment mode of Table 3, and the specific genotype of mutation site is determined.

[0063] Comparison group:

[0064] According to the reaction system as shown in Table 2, the above genomic DNA samples are directly subjected to fluorescent PCR amplification, under thermal cycling conditions, first denature at 95℃ for 10 minutes, then 40 cycles, each cycle includes 95℃ denaturation for 15 seconds and 60℃ annealing + extension for 1 minute, during which the fluorescence signal is collected. According to the fluorescence amplification curve, the Ct values of FAM signal, VIC signal and ROX signal are obtained respectively, and whether each position exists mutation site is determined according to the judgment mode of Table 3, and the specific genotype of mutation site is determined.

[0065] 2. Specificity analysis

[0066] Detection: The above normal sample, mutant sample 1, mutant sample 2, mutant sample 3 and mutant sample 4 are detected by the above experimental group and comparative group respectively. The normal sample can be used as a negative sample, and the mutant sample 1, mutant sample 2, mutant sample 3 and mutant sample 4 can be used as a positive sample. If specific fluorescent signals can be detected in the positive sample, and no fluorescent signals can be detected in the negative control sample (Ct value is greater than the set threshold value), it indicates that the method has good specificity and can accurately distinguish samples containing the target mutation site from samples not containing the site. If fluorescent signals are detected in the negative control sample (false positive), it indicates that the specificity is poor.

[0067] Table 4 experimental group detection results

[0068] Sample type FAM Ct value VIC Ct value ROX Ct value Normal sample 22.3 24.1 23.8 Mutant sample 1 No Ct value 25.9 24.7 Mutant sample 2 25.5 No Ct value 25.1 Mutant sample 3 25.4 26.3 No Ct value Mutant sample 4 No Ct value No Ct value No Ct value

[0069] As shown in Table 4, the detection results of the experimental group on the normal sample, mutant sample 1, mutant sample 2, mutant sample 3 and mutant sample 4 are as expected, indicating that the experimental group has very high specificity.

[0070] Table 5 comparative group detection results

[0071] Sample type FAM Ct value VIC Ct value ROX Ct value Normal control 25.4 26.5 25.1 Mutant sample 1 No signal 35.2 31.5 Mutant sample 2 25.3 46.2 25.4 Mutant sample 3 25.2 26.1 46.9 Mutant sample 4 No Ct value 48.3 49.2

[0072] As shown in Table 5, the detection results of the comparative group on the normal sample and mutant sample 1 are as expected, while the comparative group on mutant sample 2, mutant sample 3 and mutant sample 4 respectively appears non-specific product in VIC and ROX signal channels, and the fluorescent signal is strong, resulting in high Ct value, indicating that the specificity of the comparative group is not as good as that of the experimental group.

[0073] 3. Sensitivity analysis

[0074] Mutant sample 4 is diluted and prepared to obtain 1 copy / μL, 10 copies / μL, 100 copies / μL, 1000 copies / μL, 10000 copies / μL, 100000 copies / μL and 1000000 copies / μL, to obtain DNA samples of different concentrations. The experimental group and the comparative group are used to detect these different concentrations of samples. Record the lowest DNA concentration that can detect positive signals (fluorescent signals exceeding threshold value). The lower the lowest DNA concentration that can detect positive signals, the higher the sensitivity.

[0075] Table 6 comparative group detection results

[0076] Sample concentration (copy number / μL) Experimental group Comparison group 1000000 Positive Positive 100000 Positive Positive 10000 Positive Positive 1000 Positive Positive 100 Positive Positive 10 Positive Negative 1 Positive Negative

[0077] As shown in Table 6, the experimental group method can detect the lowest DNA concentration of 1 copy number / μL, while the comparative group method can detect the lowest DNA concentration of 102 Copy number / μL. This indicates that the experimental group method is 100 times more sensitive than the comparative example, and can detect lower concentrations of DNA samples containing mutation sites, with a significant advantage in detecting trace mutations.

[0078] 1. Sample

[0079] Two groups of subjects were selected for comparative analysis: the ADHD patient group included 300 children and adolescents aged 6 to 18 years old, all of whom met the detection criteria of DSM-5; the control group selected 300 healthy individuals matched in age and gender with the ADHD patient group, and these healthy control group members had no history of neuropsychiatric diseases.

[0080] 2. Three-site combination detection steps

[0081] Sample preparation: 2-5 mL of peripheral blood was collected from the subjects, and EDTA was used for anticoagulation. QIAamp DNA Blood MiniKit (250) (QIAGEN) was used to extract genomic DNA. NanoDrop was used to determine the DNA concentration, which was adjusted to 50 ng / μL. The A260 / A280 ratio of the resulting sample was 1.8-2.0, and the A260 / A230 ratio was ≥2.0. The sample was cut using the target cleavage reaction system of Experimental Group 1 described above, and the resulting cleavage products were reacted using the fluorescent PCR reaction system shown in Table 2. If the fluorescent PCR results of the three sites are all Ct values or Ct>35, and the subject is an ADHD patient, then the sample is a true positive sample. If the fluorescent PCR results of the three sites are all Ct values or Ct>35, but the subject is not an ADHD patient, then the sample is a false positive sample. If the fluorescent PCR results of the three sites are all Ct<30, and the subject is not an ADHD patient, then the sample is a true negative sample. If the fluorescent PCR results of the three sites are all Ct<30, but the subject is an ADHD patient, then the sample is a false negative sample.

[0082] 3. Single-site detection steps

[0083] The same sample processing and target cleavage method described above was used to obtain the cleavage products, which were amplified by fluorescent PCR in the manner shown in Table 2 (only one set of primers and probes was added to detect the corresponding mutation site). If the fluorescent PCR result of a single site is a Ct value or Ct>35, and the subject is an ADHD patient, then the sample is a true positive sample. If the fluorescent PCR result of a single site is a Ct value or Ct>35, but the subject is not an ADHD patient, then the sample is a false positive sample. If the fluorescent PCR result of a single site is Ct<30, and the subject is not an ADHD patient, then the sample is a true negative sample. If the fluorescent PCR result of a single site is Ct<30, but the subject is an ADHD patient, then the sample is a false negative sample.

[0084] Detection results

[0085] Table 7 Statistical results of detection data of each group

[0086] Detection method ADHD positive number / total number Healthy control false positive number / total number Sensitivity Specificity Site 1 detection alone 216 / 300(72%) 42 / 300(14%) 72% 86% Site 2 detection alone 219 / 300(73%) 39 / 300(13%) 73% 87% Site 3 detection alone 204 / 300(68%) 48 / 300(16%) 68% 84% Three-site combined detection 279 / 300(93%) 15 / 300(5%) 93% 95%

[0087] As shown in Table 7, three-site combination detection significantly improves the sensitivity and specificity of ADHD diagnosis, reaching 93% and 95% respectively, which is better than single-site detection. This indicates that multi-site joint detection can more accurately identify ADHD patients and reduce misdiagnosis rate, providing more reliable basis for clinical diagnosis.

[0088] Table 8 ROC curve parameters of each group

[0089] Detection method AUC 95% confidence interval P value (vs combination) Site 1 detection alone 0.7109 0.5478-0.8739 <0.001 Site 2 detection alone 0.7063 0.5369-0.8758 <0.001 Site 3 detection alone 0.6247 0.4525-0.7970 <0.001 Three-site combined detection 0.8968 0.7794-1.0000 -

[0090] For Figure 3 The ROC curve parameters of each group were statistically analyzed, as shown in Table 8. The AUC value of three-site combination detection was significantly higher than that of single-site detection, further verifying its superiority, with a wider confidence interval and P value less than 0.001, indicating that the difference has statistical significance.

[0091] Table 9 Clinical subgroup analysis of each group

[0092] ADHD subtype Site 1 positive rate Site 2 positive rate Site 3 positive rate Combined positive rate Attention deficit type (n=120) 65% 58% 42% 88% Hyperkinetic impulsive type (n=80) 52% 68% 48% 90% Mixed type (n=100) 71% 73% 62% 95%

[0093] As shown in Table 9, three-site combination detection showed high positive rate in different ADHD subtypes, especially in mixed type up to 95%, significantly better than single-site detection, further confirming its comprehensiveness and effectiveness in clinical diagnosis. In addition, the high positive rate of combination detection in different subtypes indicates that it can more accurately identify the diversity of ADHD, and combination detection can identify such complex mutation individuals, reflecting the threshold effect.

[0094] In terms of detection efficiency, the sensitivity of combination detection is improved by 20%-25% compared with single-site detection, the misdiagnosis rate is reduced from 27%-32% to 7%, and the specificity is improved from 84%-87% to 95%.

[0095] In terms of accurate identification of disease subtypes, the positive rate of combination detection for attention deficit patients (88%) is significantly higher than that of site 3 alone (42%), and the positive rate for hyperactive-impulsive patients (90%) is significantly higher than that of site 1 alone (52%), indicating that combination detection can more comprehensively cover the genetic heterogeneity of ADHD.

[0096] In the aspect of early detection, the positive rate of the combined detection (82%) is significantly higher than that of the single site detection (the highest 58%) in the patients with mild ADHD. For the high-risk children under 6 years old, the proportion of the positive children of the combined detection diagnosed with ADHD within 2 years is 80%, while that of the positive children of the single site detection is only 33%. This shows that the combined detection has obvious advantages in early detection.

[0097] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A kit for detecting a neurodevelopmental disorder disease, characterized by, The CRISPR / Cas9 cutting system and the fluorescent PCR amplification system; wherein the CRISPR / Cas9 cutting system comprises sgRNA-1 as shown in SEQ ID NO. 2, sgRNA-2 as shown in SEQ ID NO. 3, sgRNA-3 as shown in SEQ ID NO. 4 and Cas9 protein; the fluorescent PCR amplification system comprises DNA molecules as shown in SEQ ID NO. 5-13, the CRISPR / Cas9 cutting system is used for targeted cutting of genomic DNA to obtain a target sequence comprising a SCN1A gene multi-mutation site, the fluorescent PCR amplification system is used for fluorescent amplification of the target sequence, the SCN1A gene multi-mutation site is a single nucleotide deletion mutation at chr2: 165984651, chr2: 165984876 and chr2: 165984932, the reference genome is GRCh38.p14, and the neurodevelopmental disorder is attention deficit hyperactivity disorder; the DNA as shown in SEQ ID NO. 5 and the DNA as shown in SEQ ID NO. 6 are a primer pair, and the DNA as shown in SEQ ID NO. 7 is a TaqMan probe; the DNA as shown in SEQ ID NO. 8 and the DNA as shown in SEQ ID NO. 9 are a primer pair, and the DNA as shown in SEQ ID NO. 10 is a TaqMan probe; the DNA as shown in SEQ ID NO. 11 and the DNA as shown in SEQ ID NO. 12 are a primer pair, and the DNA as shown in SEQ ID NO. 13 is a TaqMan probe.

2. The kit of claim 1, wherein The CRISPR / Cas9 cutting system specifically comprises Cas9 protein with a final concentration of 50-200 nM, sgRNA-1 with a final concentration of 50-300 nM, sgRNA-2 with a final concentration of 50-300 nM, sgRNA-3 with a final concentration of 50-300 nM, 50-200 ng of genomic DNA, 10x NE buffer 3.1, BSA with a final concentration of 0.05-0.15 mg / mL, RNase inhibitor with a final concentration of 0.1-1 U / μL, and double distilled water.

3. The kit of claim 2, wherein The CRISPR / Cas9 cutting system specifically comprises Cas9 protein with a final concentration of 100 nM, sgRNA-1 with a final concentration of 200 nM, sgRNA-2 with a final concentration of 200 nM, sgRNA-3 with a final concentration of 200 nM, 100 ng of genomic DNA, 10x NE buffer 3.1, BSA with a final concentration of 0.1 mg / mL, RNase inhibitor with a final concentration of 0.5 U / μL, and double distilled water.

4. The kit of claim 1, wherein The fluorescent PCR amplification system comprises 5-10 ng of the cleavage product DNA, 12.5 μL of 2x TaqMan Master Mix, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 5, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 6, 0.2 μM of the DNA molecule as shown in SEQ ID NO. 7, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 8, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 9, 0.2 μM of the DNA molecule as shown in SEQ ID NO. 10, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 11, 0.4 μM of the DNA molecule as shown in SEQ ID NO. 12, 0.2 μM of the DNA molecule as shown in SEQ ID NO. 13, and double distilled water, in a total volume of 100 μL.

5. The kit of claim 1, wherein A triphosphate label is added to the 5' end of sgRNA-1 as shown in SEQ ID NO. 2, a triphosphate label is added to the 5' end of sgRNA-2 as shown in SEQ ID NO. 3, and a triphosphate label is added to the 5' end of sgRNA-3 as shown in SEQ ID NO.

4.

6. Use of the kit of any one of claims 1 to 5 in the preparation of a kit for detecting attention deficit hyperactivity disorder.

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