Primer probe composition and application thereof

Abstract

The invention relates to the technical field of gene detection, and discloses a primer probe composition and application thereof.The primer probe composition comprises a primer group and a probe group, and the primer group comprises a first primer pair F1, R1 and R2; a second primer pair F2 and R3 and a third primer pair IC-F and IC-R; the probe group comprises a first probe P1, a first probe P2, a second probe P3 and a third probe IC-P. The primer probe composition can accurately detect the copy number of SMN1 and SMN2, the detection accuracy is higher than that of a conventional kit in the market, and the detection linearity is good. By matching with a digital PCR technology, the detection LOB value is 2 copies / reaction, and the LOD value is 20 copies / [mu] L of reaction liquid. The method can be applied to preparation of high-accuracy SMA detection kits or preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer-probe combination and application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Spinal muscular atrophy (SMA) is a group of hereditary neuromuscular diseases with muscle weakness and atrophy as the main clinical features caused by degeneration of α-motor neurons in the anterior horn of the spinal cord. It is the most common neuromuscular disease in children. SMA is an autosomal recessive genetic disease with a carrier rate of 1 / 50~1 / 40 and an incidence rate of 1 / 10000~1 / 6000 live births. If both parents are carriers, the probability of the newborn being sick is 1 / 4.

[0004] SMA is associated with the SMN survival of motor neurons gene. SMN has two highly homologous copies, SMN1 and SMN2, which differ by only 5 bases, 2 of which are located in exons 7 and 8, and the other 3 bases are in introns 6 and 7. Homozygous deletion of exon 7 of the SMN1 gene is the main cause of SMA. In healthy people, this gene produces survival of motor neurons or SMN protein, which is essential for nerve function to control muscles. If SMN1 exon 7 is missing, SMN protein cannot be synthesized, causing nerve cells to be unable to function normally and eventually die, resulting in muscle weakness, which in turn affects people's daily life and even life. An increase in the number of copies of the SMN2 gene is beneficial to the alleviation of SMA symptoms. Each person may have 0, 1, 2, 3, or 4 copies of SMN2. SMN2 is currently recognized as a modifying factor for SMA. The more SMN2 copies a patient carries, the milder the phenotype. Although its correlation with the phenotype is not completely consistent, the SMN2 copy number is still used as one of the standard steps for SMA diagnosis in domestic and international management consensus.

[0005] The detection of SMN1 and SMN2 copy numbers can be used for genetic diagnosis of diseases, carrier screening, prenatal diagnosis of infants, preimplantation genetic testing, etc. It can help confirm the patient's disease type during the disease diagnosis process for accurate medication, help identify asymptomatic individuals who carry pathogenic gene mutations, thereby providing families with genetic counseling and fertility options, assisted reproduction screening to remove healthy embryos, and reduce the risk of passing genetic diseases to the next generation. It not only helps in the early diagnosis and prevention of diseases, but also provides individuals and families with important genetic reference information.

[0006] The existing technologies for detecting spinal muscular atrophy include: first, chromosome karyotype analysis, to determine whether there are abnormal chromosomes; mitochondrial DNA detection: to determine whether there are abnormal mitochondria. These two methods are relatively subjective in judging the results and are prone to missed detection and wrong detection. Secondly, there is conventional gene mutation detection, which determines whether it is caused by gene mutation by detecting gene sequence. This method is relatively cumbersome to operate. There is also a serum detection method, which detects the content of specific enzymes in serum, but this method is an indirect detection and can only assist in determining whether there is spinal muscular atrophy; the quantification of SMN gene copy number has become the current mainstream method, including DHPLC and other technologies. The principle of this method is that after DNA is denatured and gradually cooled and annealed, the heterozygous and wild-type PCR amplification products form homologous duplexes while also mismatching to form heteroduplexes. Under partial denaturation conditions, mismatched heteroduplex DNA is more likely to melt into single-stranded DNA, and the binding force with the DNA elution column is reduced. It is easier to be eluted by the eluent than homologous duplex DNA molecules, thereby separating from homologous duplex DNA. However, the disadvantage is that this method is limited by primers, reaction conditions, temperature when binding to the column and other factors, resulting in poor stability of the results.

[0007] There are currently a number of test kits on the market that use qPCR and related methods to measure exon 7 of SMN1 and SMN2, with a detection limit as low as 0.25 ng / μL. However, using qPCR and related methods for copy number detection has the problem of low accuracy of the results. Summary of the invention

[0008] The purpose of the present invention is to provide a primer probe combination and a kit using the same in view of the deficiencies of the current prior art. The probe and primer combination of the present invention are designed for special sites of SMN1 and SMN2. The site is located at the differential base (c.840C>T) in the coding region of exon 7 and the differential base (c.239G / A) in the coding region of exon 8 for detection, and the special design of the primer probe makes the binding ability of the primer probe stronger, and the selection of the primer and probe sequence fragments makes the primer and probe less likely to form a loop structure, so as to achieve the purpose of absolute quantification of SMN1 and SMN2 genes, and the quantitative results are stable and reliable.

[0009] The technical solution of the present invention is as follows: A primer-probe combination, comprising a primer set and a probe set, The primer set includes a first primer pair complementary to the SMA pathogenic gene region, a second primer pair, and a third primer pair complementary to the intracellular housekeeping gene; the sequence of the first primer pair is F1, R1, R2 as shown in SEQ ID NO.1-3, the sequence of the second primer pair is F2, R3 as shown in SEQ ID NO.6 and SEQ ID NO.7, and the sequence of the third primer pair is IC-F, IC-R as shown in SEQ ID NO.9 and SEQ ID NO.10; The probe group includes a first probe complementary to the SMA pathogenic gene region, a second probe, and a third probe complementary to the intracellular housekeeping gene; the first probe is P1 and P2 as shown in SEQ ID NO.4 and SEQ ID NO.5, the sequence of the second probe is P3 as shown in SEQ ID NO.8, and the sequence of the third probe is IC-P as shown in SEQ ID NO.11.

[0010] Preferably, the first probe, the second probe and the third probe are respectively labeled with different fluorescent markers, wherein the fluorescent markers are selected from fluorescent dyes with maximum emission wavelengths of 518 nm, 538-555 nm, 574-575 nm, 602-615 nm, 640 nm, 660-667 nm or 690 nm.

[0011] Preferably, the first primer pair and the first probe specifically amplify the differential base (c.840C>T) region of SMN1 exon 7; the second primer pair and the second probe specifically amplify the differential gene (c.239G / A) region of SMN1 exon 8. The first primer pair is designed so that the 5' end portion of the first probe (the base to the left of the middle modified base) contains 16 bases, which do not have a complementary sequence to the target sequence of the amplified product, but are complementary to the sequence at the 5' end of the corresponding upstream primer, the 3' end portion of the probe (the base to the right of the middle modified base) contains 16 bases, which are complementary to the target sequence of the amplified product, and the 5' end of the upstream primer contains a sequence that is not paired with the target sequence of the amplified product, but is partially complementary to the 5' end of the probe. In order to improve the specificity of the probe, the region complementary to the template chain at the 3' end of the probe is designed to be relatively short. When the upstream and downstream primers bind to the template to amplify and form products, the probe specifically binds to the completely complementary template product with the help of the 5' end binding to the complementary chain of the upstream primer, while the mismatched product does not bind, thereby distinguishing the mutant types in the (c.840C>T) region and the (c.239G / A) region.

[0012] Another aspect of the present invention provides use of the primer-probe combination as described above in the preparation of a spinal muscular atrophy (SMA) gene mutation detection preparation and / or kit.

[0013] A spinal muscular atrophy (SMA) gene mutation detection kit comprises the primer-probe combination as described above.

[0014] According to a preferred embodiment, it also includes a universal digital PCR reaction premix (DNA, 2×), an enhancer, a blank control, a negative control, and a positive control.

[0015] Another aspect of the present invention provides the steps of using the primer-probe combination as described above to prepare a spinal muscular atrophy (SMA) gene mutation detection kit for spinal muscular atrophy (SMA) gene mutation detection: Step (1) obtaining a biological sample from a test subject; Step (2) uses the probe and primer combination to perform fluorescence quantitative PCR on the biological sample to determine whether there is a spinal muscular atrophy (SMA) mutant gene.

[0016] According to a preferred embodiment, the biological sample is selected from venous blood.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. A primer-probe combination and its application, which can absolutely quantify the copy number of a gene, obtain the relative copy number of SMN1 and SMN2 by comparing with the internal reference gene, and avoid the problem of missed detection due to integration loss to the greatest extent; and through the primer-probe design of specific sites and the addition of enhancers, SMN1 and SMN2 can be effectively distinguished, solving the problem of non-specific amplification caused by the high homology of the two genes; 2. A primer-probe combination and its application: ① High sensitivity: it can detect extremely small amounts of nucleic acid samples under complex backgrounds; ② Strong specificity: it can effectively distinguish between SMN1 and SMN2 genes, and detect SMN1 without interference from the high background of SMN2; ③ Absolute quantification: the test sample can be absolutely quantified without the need for standards; ④ Low sample demand, and multiple specimen types can be detected; ⑤ High tolerance: it reduces the impact between reaction systems and the interference of background sequences and inhibitors on the reaction. These changes have improved the detection sensitivity of this project and reduced the probability of missed detection and false positives. ⑥ Dual-gene detection: In addition to detecting the SMN1 gene, digital PCR can also detect the copy number of the SMN2 gene, providing more information for disease diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the linear regression equation of Example 2 of the present application; Figure 2 SD, CV and adv. results of Example 2 of the present application; Figure 3The data statistics of the NC group and the PC group for the determination of the LOD performance in Example 3 of the present application; Figure 4 This is a statistical diagram of the data of the NC group and the PC group for the precision determination in Example 4 of the present application; Figure 5 This is a test data result diagram of the thermal stability test in the reagent stability test of Example 5 of the present application; Figure 6 This is a graph of the test data results of the freeze-thaw stability test in the reagent stability test of Example 5 of the present application. DETAILED DESCRIPTION

[0019] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0021] In people with normal phenotypes (including normal people and carriers), the number of SMN1 gene copies is usually 1 to 4. The normal human genotype includes the common [1+1] type and the rare [2+1] type and [2+2] type. In carriers, one chromosome contains 1 or 2 copies of a functional SMN1 gene, and the other chromosome contains a functionally abnormal SMN1 gene with a deletion or slight variation. The genotype can be a heterozygous deletion ([1+0] type, [2+0] type) or a heterozygous mutation ([1+1d] and [2+1d] type) [d represents a slight variation in the SMN1 gene and abnormal function, such as a nonsense mutation, frameshift mutation, missense mutation, etc.]. Among SMA patients with abnormal phenotypes, there are two main types of mutant genotypes: 95% are caused by homozygous deletion of SMN1 biallelic genes ([0+0] genotype); 5% are caused by compound heterozygous mutations of SMN1 ([0+1d] genotype); and it is very rare for both SMN1 biallelic genes to be minor mutations ([1d+1d]). Most SMN1 deletions are exon 7 and exon 8 deletions, and a small number are exon 7 deletions only.

[0022] The copy number of SMN2 ranges from 0 to multiple. The copy number of SMN2 is currently recognized as a modifier of SMA. The more copies of SMN2 a patient carries, the milder the phenotype, but its correlation with the phenotype is not completely consistent. Sequence variations of SMN2 and other genes may also affect the phenotype of SMA. Therefore, the copy number results of SMN2 can only provide a possible reference information for the clinical severity of children or fetuses with SMA rather than a definitive conclusion. In addition, the copy number of SMN2 does not need to be tested for non-patient populations. It should also be noted that both SMN1 and SMN2 are located at 5q13.2. There are multiple pairs of homologous genes in this region, including SMN, which lead to unequal exchange and gene conversion of the genome, resulting in various changes in the copy number of SMN1 and SMN2.

[0023] Since the SMA pathogenic genes SMN1 and SMN2 are DNA, the invention adopts the polymerase chain reaction method with end-point fluorescence monitoring and the designed primer-probe combination to detect the differential base sites (c.840C>T) located in the coding region of exon 7 and the differential base sites (c.239G / A) located in the coding region of exon 8, to absolutely quantify SMN1 and SMN2, and to obtain the relative copy numbers of SMN1 and SMN2 by comparing with the internal reference gene.

[0024] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0025] Example 1 The primer-probe combination of the present invention is used to perform SMA detection to determine the detection accuracy of the primer-probe of the present application.

[0026] The primer-probe combination sequence included in the present invention is as shown in the following sequence table:

[0027] The components of the kit included in the present invention are as follows:

[0028] The following are the performance test indicators for the PCR amplification reagent of the present invention: Blank limit: When testing clinical negative samples, the detection result is negative.

[0029] Limit of Detection (LOD): The lowest concentration that can be detected by the test method. In this test, a positive detection rate of ≥95% (19 / 20) is used as the standard for determining LOD.

[0030] Precision: The precision reference material was tested and the intra-batch precision met the coefficient of variation of the concentration value (CV, %) ≤ 10.0%.

[0031] Linearity: Sample gradient dilution amplification, linear regression R 2 ≥0.98 is used as the qualification standard for evaluating reagents.

[0032] Stability: heat accelerated stability, freeze-thaw stability.

[0033]

[0034] LOB performance measurement scheme (1) One batch of reagents is used to test 20 negative samples (blank control); (2) Standard: Sort the experimental results from small to large and take the 95% value as the LOB value; (3) Instruments and equipment: MicroBio D600 digital PCR analysis system.

[0035] Test results:

[0036] in conclusion (1) The blank control was tested 24 times, of which 13 groups of false positive droplets appeared in the FAM channel (SMN1exon7), 12 groups of false positive droplets appeared in the HEX channel (RPPH1), 11 groups of false positive droplets appeared in the ROX channel (SMN2exon7), and 9 groups of false positive droplets appeared in the Cy5 channel (SMN1exon8), with 1-2 false positive droplets in each reaction; (2) Conclusion: According to the test results, the LOB value is 2 copies / reaction, which means that the blank control has no more than 2 positive droplets per reaction.

[0037] Example 2 Linear Range Performance Verification Program

[0038] Specific steps: (1) Template selection: Select human genome standards with normal genotype as test samples (2) Determination of stock solution concentration: Take the high-concentration template to be measured, dilute it 10 times, measure the concentrations of two groups of templates with different concentrations, dilute the high-concentration template to 10,000 copies / μL according to the measured value, and then start 2.5-fold gradient dilution, dilute 8 gradients, and the dilution is as shown in the following table;

[0039] (3) Linear range determination: Add 2.5ul of sample to every 25ul of reaction system of the diluted template for determination, and perform 3 replicates for each concentration gradient. (4) Analysis of measurement results: Take the average value of the measurement results and perform linear analysis; (5) Instruments and equipment: MicroBio D600 digital PCR analysis system result

[0040] The data of the replicate groups were averaged to obtain the following data:

[0041] Perform linear calculation on the above data and obtain the linear regression equation as follows: Figure 1 The SD, CV and adv are calculated for the test data. The results are shown in Figure 2 shown.

[0042] in conclusion (1) According to the data, the linear R 2 They are 0.9988, 0.9982, 0.9986 and 0.9988 respectively, all greater than or equal to 0.99, indicating that the primer-probe combination has good detection linearity within the concentration range of 1.64 copies / μL-1000.00 copies / μL.

[0043] (2) According to the data display (CV heat map), it can be preliminarily determined that the LOD of the system for detecting RCNs is 25.6 copies / μL reaction solution, that is, 625±100 copies / reaction, which provides a reference for the next LOD test.

[0044] Example 3 Determination of LOD performance plan

[0045] (1) Assay template preparation: Select the positive and negative controls diluted to 500 copies / μL in the linearity test and perform LOD tests respectively; (2) LOD pre-experiment: According to the linear results of Example 2, samples with 300, 400, and 500 copies / reaction were selected for 8 repeated experiments. If the RCNs of the sample can be accurately determined in all 8 experiments, and the quantitative CV values ​​of each channel are within 10%, then this sample can be used for formal LOD determination; if none of the three concentration groups meets the above requirements, the concentration can be increased by 100 copies for formal LOD determination. Finally, it was determined through the pre-experiment that 500 copies / reaction met the test standards and the formal LOD test could be carried out.

[0046] (3) LOD determination: 1.25 μL of sample was added to every 25 μL of reaction system for determination. Each set of templates was repeated 20-24 times. The final positive detection rate of ≥95% (23 / 24) was used as the standard for determining LOD. (The detection standard for NC quality control products is that the RCNs of exons 7 and 8 (i.e., S1E7 and S1E8) of SMN1 are both 2 copies, and SMN2 is also 2 copies; the positive detection standard for PC quality control products is that the RCNs of exons 7 and 8 (i.e., S1E7 and S1E8) of SMN1 are both 0 copies, and SMN2 is multiple copies (>2 copies, theoretically 4 copies)) (4) Instruments and equipment: MicroBio D600 digital PCR analysis system result

[0047]

[0048] The statistical diagram of the NC group and PC group is shown in Figure 3 shown.

[0049] result (1) The SMN copy number variation detection reagent was used to perform 24 repeated tests on two sets of quality control products with a concentration of 20 copies / μL reaction solution. The detection rates were 100% and 100%, respectively, which met the LOD determination standard.

[0050] However, the quantitative deviation of the SMN2 relative copy number of the PC quality control product from the theoretical value is large (lower). Considering that the quality control product is an artificial plasmid synthesis template, it contains a large number of repetitive sequences in the design and is difficult to synthesize, which may lead to low template quality. In addition, the samples need to be digested by enzymes and diluted, resulting in large deviations in the relative copy numbers between targets.

[0051] (2) Conclusion: According to the test results, LOD = 20 copies / μL reaction solution (i.e. 500 copies / reaction).

[0052] Example 4 Determination of Precision plan (1) Assay template preparation: Quantify two sets of templates with different genotypes (NC and PC), generally with a concentration of 10 3 -10 4 The template of 1500 copies / μL was diluted to 1500 copies / μL according to the actual quantitative results and used as the template for precision determination. (2) Precision measurement: 1500 copies / μL of template was added to 2 μL of sample per 25 μL reaction system, i.e. 120 copies / μL reaction solution (3000 copies / reaction). Each group of templates was repeated 12 times. The coefficient of variation (CV, %) of the template quantitative detection result ≤ 10.0% was used as the standard for evaluating the precision of the kit.

[0053] (3) Instruments and equipment: MicroBio D600 digital PCR analysis system result:

[0054]

[0055] The statistical diagram of the NC group and PC group is shown in Figure 4 shown.

[0056] in conclusion (1) The absolute quantification and RCNs CV values ​​of each group of templates in 2000-3000 copies / reaction were less than 5%, which was within the acceptable range; (2) This reagent has good precision performance.

[0057] Example 5 Reagent Stability plan

[0058] Specific steps: (1) Reagent preparation: The reagents that need to be accelerated include 2X mix, 10X primer probe mix and Taq enzyme. Each experiment requires 12 times. Heat acceleration is performed according to the packaging specifications. Since the minimum specification is 24 times, 3 batches of experimental reagents with a specification of 24 times are prepared; (2) Reagent packaging: Pack the prepared reagents into 3 storage tubes provided by Michael, with each reagent having a volume for 24 experiments, as follows:

[0059] (3) Thermal acceleration: Place the three reagents in small ziplock bags according to the table below, place them in a 37°C water bath according to the specific date, and store them under heating. Store the other reagents in a -20°C refrigerator.

[0060] (4) Freeze-thaw experiment: According to the table below, place the three reagents in small ziplock bags and conduct freeze-thaw experiments on specific dates. Store the other reagents in a -20°C refrigerator. The freeze-thaw method is to place them at room temperature for a certain period of time to fully thaw, and then place them in a -20°C refrigerator overnight to fully freeze;

[0061] (5) Thermal acceleration test verification: Take three batches of reagents that have been accelerated and add a control reagent, and perform the test according to the following method; First, conduct experiments with reagents stored at -20℃ and accelerated at 37℃ for 3 days and 5 days. If the experimental data of thermal acceleration for 3 days and 5 days are consistent with the results of the reagents stored at -20℃, the thermal acceleration for 7 days can be conducted. If the data of thermal acceleration for 5 days is problematic, the thermal acceleration for 7 days can be discontinued. (6) Freeze-thaw experiment verification: Take the three batches of reagents that have been accelerated and add the control reagent, and perform the measurement according to the following method; First, conduct a freeze-thaw experiment for 5 days, 7 days, and a -20°C control system. If the results of freeze-thaw for 5 days and 7 days are no different from those of the -20°C control system, the freeze-thaw experiment for 3 days can be skipped. If the results of freeze-thaw for 5 days and 7 days are problematic, the freeze-thaw experiment for 3 days can be repeated. result (1) Thermal stability test (copies / μL_reaction solution, 25μL)

[0062] Test results such as Figure 5 shown.

[0063] (2) Freeze-thaw stability test (copies / μL_reaction solution, 25μL)

[0064] Test results such as Figure 6 As shown ( Figure 6 The horizontal and vertical coordinates of Figure 5 in the same order as in ).

[0065] in conclusion (1) Thermal stability The reagent was incubated at 37°C for 3 / 5 / 7 days, and the minimum detection limit of NC and PC was 100%; The quantitative values ​​of each target gene after the reagent was incubated at 37°C for 3 / 5 / 7 days were consistent with those of the control group.

[0066] This reagent has good thermal stability and can be transported at room temperature for a short time.

[0067] (2) Freeze-thaw stability The minimum detection limit of NC and PC detection rates were both 100% after the reagents were frozen and thawed 3 / 5 / 7 times; The quantification of the internal reference gene and target gene after the reagent was frozen and thawed 7 times was consistent with that of the control group.

[0068] This reagent has good freeze-thaw stability.

[0069]

[0070] Example 6 A probe and primer combination A probe and primer combination comprising the following components: (1) A probe set, comprising a first probe complementary to a spinal muscular atrophy (SMA) mutant gene region, the sequences of which are P1 and P2 as shown in SEQ ID NO.4 and SEQ ID NO.5; a second probe complementary to a spinal muscular atrophy (SMA) mutant gene region, the sequence of which is P3 as shown in SEQ ID NO.8; and a third probe complementary to DNA of a housekeeping gene in a cell, the sequence of which is IC-P as shown in SEQ ID NO.11; (2) A primer set, comprising a first primer pair complementary to a spinal muscular atrophy (SMA) mutant gene region, the sequences of which are F1, R1, and R2 as shown in SEQ ID NOs. 1-3; a second primer pair complementary to a spinal muscular atrophy (SMA) mutant gene region, the sequences of which are F2 and R3 as shown in SEQ ID NOs. 6 and 7; and a third primer pair complementary to a housekeeping gene, the sequences of which are IC-F and IC-R as shown in SEQ ID NOs. 9 and 10.

[0071] The first probe is labeled with a fluorescent dye with a maximum emission wavelength of 522 nm, the second probe is labeled with a fluorescent dye with a maximum emission wavelength of 602 nm, and the third probe is labeled with a fluorescent dye with a maximum emission wavelength of 664 nm.

[0072] The first probe and the first primer pair are used to amplify the template of the spinal muscular atrophy (SMA) mutant gene region on ABI7500; the second probe and the second primer pair are used to amplify the template of the spinal muscular atrophy (SMA) mutant gene region on ABI7500; the third probe and the third primer pair are used to amplify the template of the intracellular housekeeping gene on ABI7500. Both the probe set and the primer set of the present invention can effectively amplify the relevant nucleotide sequence.

[0073] Example 7 A kit for detecting a spinal muscular atrophy (SMA) mutant gene region, comprising the probe set and primer set described in Example 6.

[0074] The kit was used to amplify the mixture of spinal muscular atrophy (SMA) mutant gene region template and intracellular housekeeping gene template on ABI7500. It can be seen that the kit can detect the gene mutation in the spinal muscular atrophy (SMA) mutant gene region and the intracellular housekeeping gene template at the same time. The intracellular housekeeping gene is used to evaluate sample quality and PCR inhibitory factors.

[0075] The positive control of the kit is a mixed plasmid of the mutant gene region template and the intracellular housekeeping gene template of patients clinically diagnosed with spinal muscular atrophy (SMA); the negative control is water.

[0076] Example 8 In order to detect the accuracy and effectiveness of the kit described in Example 1 of the present invention for diagnosing spinal muscular atrophy (SMA), two existing commercially available kits of the same type (hereinafter referred to as Kit A (Wuseshi) and Kit B (Tianlong) respectively) and the kit of the present invention (hereinafter referred to as "the present invention") were used to conduct a comparative test on 100 clinical samples to evaluate the consistency of the test system and the control system. 3.1 (1) Nucleic acid preparation Nucleic acid extraction can generally be divided into four steps: lysis-binding-washing-elution (2) Nucleic acid amplification (digital PCR) Nucleic acid amplification can generally be divided into: PCR amplification reagent preparation (reagent preparation area) - nucleic acid sample loading (sample preparation area) - droplet preparation and PCR amplification (amplification area) (3) Data analysis 3.2 Sample processing (carried out in the sample processing area)

[0078] (1) Sample nucleic acid extraction The sample is a venous blood sample; the negative control sample should participate in the parallel extraction step of nucleic acid.

[0079] ① Take 10μL of pretreatment solution, put it into a 1.5mL EP tube, add 400μL of clinical sample, mix well. Then add 400μL Lysis buffer and 8μL magnetic beads, mix well, let stand for 10 minutes, and centrifuge instantly.

[0080] ② Place the EP tube on the magnetic rack for 2 minutes and discard the supernatant.

[0081] ③Add 750μL Wash buffer I and 50μL Wash buffer II, mix well, and centrifuge instantly.

[0082] ④ Place the EP tube on the magnetic rack for 2 minutes, discard the supernatant, and centrifuge instantly.

[0083] ⑤ Place the EP tube on the magnetic rack for 2 minutes, discard the residual liquid, and dry it for 2 minutes.

[0084] ⑥Add 35μL Elution buffer, mix well, and centrifuge instantly.

[0085] ⑦ Place the EP tube in a 60℃ metal bath and incubate for 10 min.

[0086] ⑧ Place the EP tube on the magnetic stand for 2 minutes, aspirate the supernatant, and discard the EP tube containing magnetic beads.

[0087] (2) Sample mixing Take N PCR reaction tubes, add 15 μL of the PCR reaction system prepared in step 3.2 to each tube, then add 5 μL each of the positive control, the negative control obtained in step 3.2 (1), and the supernatant of the sample to be tested, cap the tubes, centrifuge briefly, and transfer to the amplification area.

[0088] 3.3PCR amplification (performed in the amplification region) (1) Place each reaction tube into the fluorescent PCR detector and set the sample name; (2) PCR reaction conditions are set according to the table below;

[0089] (3) Set the reaction volume to 20 μL; (4) Instrument detection channel selection: Select FAM, VIC, ROX, and CY5 channels. The spinal muscular atrophy (SMA) gene mutations represented by each channel are as follows:

[0090] (5) After the settings are completed, save the file and run the reaction program; after the PCR program is completed, save the results and dispose of the reaction tubes and other waste according to the PCR laboratory management rules.

[0091] (6) Quality control: The Ct values ​​of all channels of the negative control product should be zero, otherwise it indicates contamination. The FAM and ROX channels of the positive control product should have Ct ≤ 32, otherwise it indicates that the positive control product is degraded or the amplification reagent is invalid, as shown in the following table.

[0092]

[0093] 3.4 Interpretation of test results (1) Quality control standards ①Threshold: The instrument's automatic setting is 10 times the standard deviation of the fluorescence signal in 3 to 15 cycles. The principle of manual setting is that the threshold line just exceeds the highest point of the negative control curve (irregular noise line) and the fluorescence background value of the sample. Try to choose the initial stage of the exponential phase.

[0094] ②Baseline: The starting point of the baseline should avoid the first few cycles (due to the increase in signal caused by high temperature), usually between 3 and 6 cycles. The end point should avoid the area where the signal has begun to increase significantly, usually 3 to 4 cycles less than the minimum Ct value of this group. It is recommended to leave an interval of more than 8 cycles between the starting point and the end point, and then analyze the results.

[0095] ③Only when the amplification curve is a typical S-shaped one can be considered as a positive amplification, otherwise it is considered as a negative amplification. If the reaction result is in the effective area (effective identification refers to the test method and positive judgment value), the result can be further analyzed, otherwise the test is invalid.

[0096] ④Results report If the FAM signal Ct value of the test sample is ≤32, it is reported as positive for spinal muscular atrophy (SMA) gene mutation; If the ROX signal Ct value of the test sample is ≤32, it is reported as positive for spinal muscular atrophy (SMA) gene mutation; If the Ct values ​​of FAM, VIC and ROX are all zero, the report is negative; if the Ct value of the test sample is >32, it is in the gray area and needs to be retested. If the retested Ct value is zero or >32, the report is negative; if the retested Ct value is ≤32, the report is positive.

[0097] ⑤Test results The specific results are shown in the following table:

[0098] The test results show that the test results of the kit of the present invention for the detection of spinal muscular atrophy (SMA) gene mutations are consistent with the positive coincidence rate of the invention kits A and B. The sensitivity is better than that of similar inventions with two-tube detection and similar inventions without internal control quality control. The sensitivity, specificity and accuracy of the kit of the present invention are better than those of kits A and B.

[0099] Example 9 Clinical sample No. 1 was detected as a negative sample for spinal muscular atrophy (SMA) gene mutation using kits A and B, and was detected as a positive sample for spinal muscular atrophy (SMA) gene mutation using the kit of the present invention; clinical sample No. 9 was detected as a negative sample for spinal muscular atrophy (SMA) gene mutation using kits A and B, and was detected as a positive sample for spinal muscular atrophy (SMA) gene mutation using the kit of the present invention.

[0100] In order to further verify the positive results detected by the kit of the present invention, for the samples whose comparison results with kits A and B were not completely consistent (clinical samples No. 1 and No. 9), the PCR products that obtained positive signals were amplified with the L1 region gene primer probe, and the PCR products were sequenced and verified by the Sanger method with specific primers, and the sequencing was handed over to a third-party sequencing company.

[0101] PCR product sequencing results: The sequences obtained by sequencing were compared with NCBI Blast, which confirmed that all samples detected as positive by the present invention but negative by the comparison reagent were true positives and correctly typed. The Blast comparison results after sequencing clinical sample No. 1 showed that sequence No. 1 was a mutated gene fragment of the spinal muscular atrophy (SMA) gene; the Blast comparison results after sequencing clinical sample No. 9 showed that sequence No. 9 was a mutated gene fragment of the spinal muscular atrophy (SMA) gene.

[0102] Combined with the above experimental results, the test results of the kit of the present invention are consistent with those of kits A and B, and the sensitivity has certain advantages, indicating that the kit of the present invention can not only detect the spinal muscular atrophy (SMA) gene mutation, but also perform quality control on the intracellular housekeeping gene detection, meet the screening requirements and provide better clinical guidance, and can largely prevent missed detection due to insufficient sensitivity or viral base mutations.

[0103] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0104] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

[0105] Primer and probe design advantages: ‌High specificity and secondary structure considerations‌: Probe design needs to be highly specific. Primer probes are designed for the specific regions of SMN1 and SMN2 to avoid nonspecific amplification due to their close homology. At the same time, amplified fragments with minimal secondary structure are selected to reduce reaction hindrance. If secondary structure cannot be avoided, the annealing temperature needs to be increased accordingly; strictly control the amplification length and GC content: the amplification length should be controlled within a certain range (such as 50-150bp) to maintain the consistency of analysis; the GC content needs to be kept in an appropriate range (such as 30%-80%) to avoid nonspecific reactions and signal interference, and avoid repetitive sequences: to ensure efficiency and repeatability, repetitive nucleotide sequences, especially continuous G, should be avoided to prevent the formation of primer dimers and hairpin structures, so as to ensure the specificity and accuracy of the reaction and avoid missed detection and false detection. Molecular beacon probes are also used in probe design. Their working principle is based on the stem-loop dual-labeled oligonucleotide probe with a hairpin structure. When paired with the template, the fluorescent group and the quenching group are separated to generate a fluorescent signal, making the signal-to-noise ratio higher.

[0106] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A primer-probe composition, characterized in that: Including primer sets and probe sets, The primer set includes a first primer pair complementary to the SMA pathogenic gene region, a second primer pair, and a third primer pair complementary to the intracellular housekeeping gene; The probe set includes a first probe complementary to the SMA pathogenic gene region, a second probe, and a third probe complementary to the intracellular housekeeping gene; The first primer pair and the first probe specifically amplify the differential base c.840C>T region of SMN1 exon 7; The second primer pair and the second probe specifically amplify the differential gene c.239G / A region of SMN1 exon 8; The first primer pair includes F1, R1, and R2, and the sequences are shown in SEQ ID NO.1-3; The second primer pair includes F2 and R3, and the sequences are shown in SEQ ID NO.6 and SEQ ID NO.7; The first probe includes probes P1 and P2, and the sequences of the probes P1 and P2 are shown in SEQ ID NO.4 and SEQ ID NO.5; The second probe is probe P3, and the sequence of the probe P3 is shown in SEQ ID NO.

8.

2. A primer-probe combination according to claim 1, characterized in that: The 5' end portion of the first probe is complementary to the sequence at the 5' end of the upstream primer of the first primer pair; the 3' end portion of the first probe is complementary to the target sequence of the amplified product; The 5' end of the second probe is complementary to the sequence at the 5' end of the upstream primer of the second primer pair; the 3' end of the second probe is complementary to the target sequence of the amplified product.

3. A primer-probe combination according to claim 1, characterized in that: The sequences of the third primer pair IC-F and IC-R are shown in SEQ ID NO.9 and SEQ ID NO.10; the sequence of the third probe IC-P is shown in SEQ ID NO.

11.

4. A primer-probe combination according to claim 3, characterized in that: The first probe, the second probe and the third probe are respectively marked with different fluorescent markers; the fluorescent markers are selected from fluorescent dyes with maximum emission wavelengths of 518nm, 538-555nm, 574-575nm, 602-615nm, 640nm, 660-667nm or 690nm.

5. Use of a primer-probe combination in the preparation of a spinal muscular atrophy (SMA) gene mutation detection preparation and / or kit, characterized in that: The primer-probe combination is the primer-probe combination according to any one of claims 1 to 3.

6. A preparation and / or kit for detecting a spinal muscular atrophy (SMA) gene mutation, characterized in that: The invention comprises a primer-probe combination as described in any one of claims 1 to 4.

7. A preparation and / or kit for detecting a spinal muscular atrophy (SMA) gene mutation according to claim 6, characterized in that: Also included is Universal Digital PCR Reaction Master Mix (DNA, 2×), Enhancer, Blank Control, Negative Control, and Positive Control.

8. A method for using a spinal muscular atrophy (SMA) gene mutation detection kit, characterized in that: The spinal muscular atrophy (SMA) gene mutation detection kit is the kit as claimed in claims 6-7, and the method of use specifically comprises the following steps: Step (1) obtaining a biological sample from a test subject; Step (2) uses the probe and primer combination to perform fluorescence quantitative PCR on the biological sample to determine whether there is a spinal muscular atrophy (SMA) mutant gene.

9. The method for using a spinal muscular atrophy (SMA) gene mutation detection kit according to claim 8, characterized in that: The biological sample is selected from venous blood.

Images