Primer group, kit and method for CGG repeated amplification disease detection

Abstract

The invention belongs to the technical field of molecular diagnosis and enzyme engineering, and particularly discloses a primer group, a kit and a method for detecting CGG repeated amplification diseases. The multiplex PCR system provided by the invention comprises a PCR primer group of which the sequence is as shown in SEQ ID NO: 1 to SEQ ID NO: 7; the DNA polymerase takes Pfu polymerase as a skeleton and comprises mutations such as V93Q, E148K, G150A, S152A or N155A and the like, and the C end of the DNA polymerase is fused with a Cren7 DNA binding protein through a short connecting peptide; and the high GC PCR system comprises LiCl, MgCl2, dNTP (deoxyribonucleoside triphosphate), 7-deaza-dGTP (7-deaza-dGTP) and the like. According to the present invention, the engineering DNA polymerase, the optimized high GC PCR system and the multiple primer group are combined, such that the detection sensitivity and the detection specificity are significantly improved, and the joint detection of a variety of clinical phenotype similar diseases is achieved.

Description

Technical Field

[0001] This invention relates to the fields of molecular diagnostics and enzyme engineering technology, and in particular to a primer set, kit, and method for detecting CGG repeat amplification diseases. Background Technology

[0002] Short tandem repeats (STRs) are widely distributed in the human genome. When their copy number exceeds a certain threshold, they can cause slippage during DNA replication, forming hairpin structures and G-quadruplexes, leading to genetic instability and disease. Among known nucleotide repeats, CGG repeat amplification can cause diseases such as Fragile X syndrome (FXS), neuronal intranuclear inclusion disease (NIID), and oculopharyngeal distal myopathy 2 (OPDM2).

[0003] FXS, caused by abnormal amplification of the CGG repeat in the 5′ untranslated region of the FMR1 gene, is the most common X-linked inherited intellectual disability. Patients typically experience onset in childhood, exhibiting intellectual disability, delayed language development, attention deficit, and autistic-like behaviors. Some cases also present with characteristic facial features and seizures. NIID, associated with CGG repeat amplification in the 5′ untranslated region of the NOTCH2NLC gene, is an adult-onset neurodegenerative disease with a complex clinical spectrum, manifesting as dementia, ataxia, Parkinson's syndrome, peripheral neuropathy, and autonomic dysfunction. The disease progresses slowly and severely impacts quality of life. OPDM2, caused by CGG (or GGC) repeat amplification in the 5′ untranslated region of the GIPC1 gene, commonly presents with oculomotor palsy, pharyngeal muscle weakness, dysphagia, and distal limb weakness. Some cases may experience respiratory involvement and elevated serum creatine kinase levels; in severe cases, muscle atrophy and respiratory failure can be life-threatening.

[0004] Although FXS, NIID, and OPDM2 are different genetic diseases, they share similar clinical manifestations. Patients may all present with cognitive decline, motor disorders, ataxia, muscle weakness, and white matter abnormalities. Imaging studies also show some overlapping features, making it difficult to differentiate them based solely on clinical symptoms and imaging results, easily leading to missed or misdiagnosis. Currently, most commonly used testing methods are single-gene tests, and except for FXS, there are no commercially available testing kits for the other diseases. Existing PCR polymerases are easily blocked by GC secondary structures when amplifying CGG repeat sequences, posing a risk of allelic bias / false ejection. Large FM in females can easily be misdiagnosed with premutated / fully mutated chimeras.

[0005] Therefore, there is an urgent need to develop new methods for detecting CGG repeat amplification diseases, enabling the detection and differentiation of FXS, NIID, and OPDM2, and avoiding misdiagnosis and missed detection due to overlapping symptoms. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention provides a primer set, kit, and method for detecting CGG repeat amplification disease. This invention utilizes engineered Pfu-mut-Cren7 polymerase and Li-containing... + A high-GC PCR system extending at ≥75℃ significantly alleviates GC secondary structure-induced stagnation and enhances the amplification capacity of ultra-long repeats. Based on this, a multiplex PCR detection kit capable of simultaneously detecting CGG repeat amplification of FMR1, NOTCH2NLC, and GIPC1 genes in the same reaction system was developed. This kit can not only cover multiple related diseases at once and improve detection efficiency, but also reduce clinical diagnostic costs and avoid misdiagnosis and missed detection due to overlapping symptoms, thus having significant application value and clinical significance.

[0007] In a first aspect, the present invention provides a set of multiplex PCR primers for the simultaneous detection of three non-coding region CGG repeat amplification diseases, namely Fragile X syndrome (FXS), intranuclear inclusion body disease (NIID), and distal ophthalmopharyngeal myopathy type 2 (OPDM2).

[0008] The primer set includes primers with sequences as shown in SEQ ID NO:1 to SEQ ID NO:7;

[0009] Among them, the 5' ends of the forward primers SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 are labeled with fluorescent groups, and any two of the fluorescent groups on the three forward primers are different.

[0010] This invention designs forward and reverse primers (SEQ ID NO: 1-6) for the CGG repeat sequences of the CGG repeat amplification genes FMR1, NOTCH2NLC, and GIPC1 in FXS, NIID, and OPDM2 diseases, respectively. Anchor primers (SEQ ID NO: 7) are then mechanically anchored to the CGG repeat regions to generate anchor primers of varying sizes. These anchor primers bind to the forward primer, producing a ladder-like peak to verify amplification. Using the multiplex PCR primer set of this invention, three target regions can be amplified simultaneously in a single reaction, enabling the detection of pathogenic CGG amplification in different diseases.

[0011] According to some embodiments of the present invention, the fluorescent groups on the three forward primers are independently selected from one of FAM, JOE, TAMRA, ROX, CY3, CY5, VIC or HEX.

[0012] In a second aspect, the present invention provides a multiplex PCR detection kit comprising components (1) to (3);

[0013] (1) The multiplex PCR primer set described in the first aspect of the present invention;

[0014] (2) Pfu-mut-Cren7 engineered DNA polymerase, wherein the DNA polymerase is based on Pfu polymerase of the archaea B family, including at least one mutation of V93Q, E148K, G150A, S152A or N155A, and the C-terminus of the Pfu polymerase is fused with Cren7 DNA binding protein via a short linker peptide.

[0015] (3) High GC PCR system, the components of which include: LiCl, MgCl2, dNTP, 7-deaza-dGTP, betaine, DMSO, propylene glycol, trehalose and nonionic surfactant.

[0016] The Pfu-mut-Cren7 engineered DNA polymerase developed in this invention is a thermostable DNA polymerase engineered through directed mutagenesis and fusion engineering. First, mutations such as V93Q, E148K, G150A, S152A, and N155A were introduced into the archaic Pfu DNA polymerase to improve its amplification performance on high-GC templates, reduce stalling caused by GC secondary structures, and enhance folding stability under high-temperature conditions. Then, a fusion design was implemented, fusing the Cren7 DNA-binding protein at the C-terminus via a short linker peptide to enhance its binding ability to the DNA template, thereby improving long-fragment amplification and resistance to inhibition.

[0017] According to some embodiments of the present invention, the components of the high GC PCR system include: 20~120mM LiCl, 1.5~3.5mM MgCl2, 8~12mM dNTP, 100~300μM 7-deaza-dGTP, 0.8~1.5M betaine, 3%~8% (v / v) DMSO, 1%~3% (v / v) propylene glycol, 0.2~0.4M trehalose, and 0.005%~0.02% (v / v) nonionic surfactant.

[0018] According to some embodiments of the present invention, the components of the high GC PCR system further include: 5~15mM KCl, 5~15mM (NH4)2SO4, and 10~30mM Tris-HCl.

[0019] According to some embodiments of the present invention, the amino acid sequence of the Pfu-mut-Cren7 engineered DNA polymerase is shown in SEQ ID NO:8.

[0020] A third aspect of the present invention provides a multiplex PCR detection method for non-disease treatment and diagnosis purposes. The method uses reagents and a sample template from the multiplex PCR detection kit described in the second aspect of the present invention, mixes them, and then performs a PCR reaction. The reaction procedure includes:

[0021] Pre-denaturation at 98℃ for 1-5 minutes;

[0022] Denaturation at 98℃ for 15-30 seconds, annealing at 58-65℃ for 30 seconds, extension at 75-78℃ for 1-3 minutes, cycle 28-35 times;

[0023] Incubate at 75~78℃ for 3~6 min to obtain the amplified product, then store at 4℃.

[0024] The extension temperature of the multiplex PCR detection method designed in this invention is set at ≥75℃ (preferably 75~78℃), and the extension time can be set to 1~3min according to the length of the amplified fragment to ensure the smooth passage of high GC template.

[0025] According to some embodiments of the present invention, the amplification products are detected by capillary electrophoresis or agarose gel electrophoresis, and the target bands or peaks of FXS, NIID and OPDM2 correspond to the CGG repeat amplification fragments of their gene non-coding regions, respectively.

[0026] A fourth aspect of the present invention provides the use of the multiplex PCR primer set as described in the first aspect of the present invention or the multiplex PCR detection kit as described in the second aspect of the present invention in the preparation of reagents for the detection of CGG repeat amplification disease.

[0027] The beneficial effects of this invention are:

[0028] Using the kit and method of this invention, the detection of three diseases, FXS, NIID, and OPDM2, can be completed simultaneously in a single reaction tube. This approach synergistically solves the problems of "difficult amplification, easy bias, and scattered detection" in CGG repeat amplification detection at three levels: enzymatic modification, system optimization, and multiple primer design. Compared with existing single-gene detection methods, this invention can not only significantly improve the amplification success rate and accuracy, but also greatly shorten the detection cycle, reduce clinical diagnostic costs, and avoid misdiagnosis and missed diagnosis due to overlapping clinical phenotypes. It has significant clinical promotion value and application prospects.

[0029] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 This is a gel electrophoresis result of DNA polymerase screening in Example 2 of the present invention;

[0032] Figure 2 This is a schematic diagram of the protein structure and corresponding amino acid sequence of the Pfu-mut-Cren7 engineered Pfu polymerase in Example 3 of the present invention;

[0033] Figure 3 This is a graph showing the detection results of silver staining of the optimized PCR system in Example 4 of the present invention;

[0034] Figure 4 This is a diagram showing the capillary electrophoresis results of the extended product of CGG repeated multiple PCR detection in Example 5 of the present invention. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] Example 1

[0038] In this embodiment, forward and reverse primers and anchoring primers were designed for the CGG repeat sequences of the FMR1 (FXS), NOTCH2NLC (NIID), and GIPC1 (OPDM2) genes, and are numbered SEQ ID NO:1-7. The primer sets are shown in Table 1:

[0039] The 5' ends of the primers FMR1-F, NOTCH2NLC-F, and GIPC1-F are labeled with the first, second, and third fluorescent groups: FAM, HEX, and TRAMA, respectively. The anchoring primers are used to randomly anchor to the CGG repeat region to generate fragments of different sizes, which can bind to the forward primers to produce a ladder-shaped peak and verify whether amplification has occurred.

[0040] The above primers are mixed to form a multiplex PCR primer set, which can simultaneously amplify three target regions in a single reaction and detect pathogenic CGG amplification in different diseases.

[0041] Example 2

[0042] This example describes the screening of DNA polymerases suitable for high-GC-PCR systems.

[0043] To identify polymerases with the potential to stably and efficiently amplify templates with high GC content for subsequent improvement and optimization, based on literature and experimental experience, 10 commonly used thermostable DNA polymerases, including Pfu, Taq, Tth, Tfl, Tfi, Tbr, Tma, Pwo, KOD, and Tli, were purchased from commercial companies or synthesized locally. At their respective optimal temperatures and systems, primer pairs SEQ ID NO:1 and SEQ ID NO:2 at a final concentration of 0.2 μM were added, along with genomic DNA from male carriers of the FMR1 gene carrying 45 CGG repeats at a template concentration of 0.4 ng / μL.

[0044] After electrophoresis of 20 μL of PCR product on a 1% agarose gel, the product concentration was analyzed by photographing. Figure 1 As shown, Pfu enzyme exhibited the best amplification effect.

[0045] Example 3

[0046] This embodiment involves sequence modification of the Pfu-mut-Cren7 engineered Pfu polymerase.

[0047] By comparing the sequence of the Pfu polymerase screened in Example 2 with other DNA polymerases with good thermostability, and using bioinformatics software such as Alphafold, amino acid sites that can improve thermostability were screened out. Point mutation sites V93Q, E148K, G150A, S152A, and N155A were screened out. Then, the corresponding amino acids on Pfu were directionally modified to improve its thermostability.

[0048] Subsequently, the C-terminus of the thermostability-modified Pfu polymerase was linked to the double-stranded DNA-binding protein Cren7, derived from extreme thermophilic archaea, to enhance its continuous synthesis capacity; at the same time, to avoid the Cren7 protein interfering with the normal protein conformation of Pfu, a linker peptide was added between the two.

[0049] The final amino acid structure and sequence of the Pfu-mut-Cren7 engineered Pfu polymerase are as follows: Figure 2As shown, the amino acid sequence of Pfu-mut-Cren7 is SEQ ID NO:8: mildvdyiteegkpvirlfkkengkfkiehdrtfrpyiyallrddskieevkkitgerhgkivrivdvekvekkflgkpitvwklylehpqdqptirekvrehpavvdifeydipfakrylidkglipmegeeelkilafdietlyhkgaeagkapiimisyadeneakvitwknidlpyvevvsseremikrflriirekdpdiivtyngdsfdfpylakraeklgikltigrdgsepkmgrigdmtavevkgrihfdlyhvitrtinlptytleavyeaifgkpkekvyadeiakawesgenlervakysmedakatyelgkeflpmeiqlsrlvgqplwdvsrsstgnlvewfllrkayernevapnkpseeeyqrrlresytggfvkepekglwenivyldfralypsiiithnvspdtlnlegcknydiapqvghkfckdipgfipsllghlleerqkiktkmketqdpiekilldyrqkaikllansfygyygyakarwyckecaesvtawgrkyielvwkeleekfgfkvlyidtdglyatipggeseeikkkalefvkyinsklpglleleyegfykrgffvtkkryavideegkvitrgleivrrdwseiaketqarvletilkhgdveeavrivkeviqklanyeippeklaiyeqitrplheykaigphvavakklaakgvkikpgmvigyivlrgdgpisnrailaeeydpkkhkydaeyyienqvlpavlrilegfgyrkedlryqktrqvgltswlnikksgthmssgkkpvkvktpagkeaelvpekvwalapkgrkgvkiglfkdpetgkyfrhklpddypi.

[0050] Example 4

[0051] This example is for optimizing the PCR system to adapt to the Pfu-mut-Cren7 engineered Pfu polymerase.

[0052] The standard PCR system is as follows: 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl and 2.0 mM MgCl2, 0.2 μM primer F / R, 10 mM dNTP, 0.4 ng / μL DNA, and 0.4 U polymerase;

[0053] The standard PCR amplification program is: 95℃ 2min → (95℃ 20s, 60℃ 30s, 72℃ 2min) × 30 cycles → 72℃ 5min.

[0054] In the optimized system of this embodiment, 50 mM LiCl, 200 µM 7-deaza-dGTP, 1 M betaine, 5% DMSO, 2% propylene glycol, 0.3 M trehalose, and 0.01% Tween-20 were additionally added.

[0055] The optimized amplification program is: 98℃ 2min → (98℃ 20s, 60℃ 30s, 75℃ 2min) × 30 cycles → 75℃ 5min.

[0056] In this embodiment, the optimized system and procedure described above were used to amplify the DNA of FXS patients carrying 200 CGG duplicates, NIID patients carrying more than 200 duplicates, and OPDM2 patients carrying 170 duplicates. The amplified bands were detected using PAGE silver staining.

[0057] The results are as follows Figure 3 As shown, bands 1, 2, 3, and 4 represent: optimized system + optimized amplification program, optimized system + normal amplification program, normal system + optimized amplification program, and normal system + normal program, respectively. The detection results show that the amplification using the optimized system + optimized amplification program in this embodiment exhibits the best amplification effect for CGG repeats.

[0058] Example 5

[0059] This embodiment uses the optimized system and optimized amplification program provided in Example 4 to perform multiplex PCR detection on CGG.

[0060] According to the optimized system and optimized amplification procedure in Example 4, DNA from male heterozygous patients with OPDM2 (170 CGG replicates), male patients with FXS (200 CGG replicates), and male patients with NIID (more than 200 CGG replicates) was used as templates, and 0.1 μM of primers SEQ NO:1~6 were added to each.

[0061] After amplification, the amplification products are detected by capillary electrophoresis, such as... Figure 4As shown, this multiplex system and amplification program effectively amplified multiple CGG repeat sites in male heterozygous patients with OPDM2 (170 CGG repeats), male patients with FXS (200 CGG repeats), and male patients with NIID (more than 200 CGG repeats). The longest CGG repeat sequence can reach more than 200 repeats. Furthermore, it can simultaneously detect the number of non-amplified repeats in normal genes, enabling the detection and differentiation of OPDM2, FXS, and NIID, avoiding misdiagnosis and missed detection due to overlapping symptoms.

[0062] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A multiplex PCR primer set for simultaneously detecting CGG repeat amplification diseases in three non-coding regions, characterized in that, The three non-coding region CGG repeat amplification diseases are Fragile X syndrome (FXS), intranuclear inclusion body disease (NIID), and distal oculopharyngeal myopathy type 2 (OPDM2). The primer set includes primers with sequences as shown in SEQ ID NO:1 to SEQ ID NO:7; Among them, the 5' ends of the forward primers SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5 are labeled with fluorescent groups, and any two of the fluorescent groups on the three forward primers are different.

2. The multiplex PCR primer set according to claim 1, characterized in that, The fluorescent groups on the three forward primers are independently selected from one of FAM, JOE, TAMRA, ROX, CY3, CY5, VIC, or HEX.

3. A multiplex PCR detection kit, characterized in that, Includes components (1) to (3), among which, (1) The multiplex PCR primer set as described in claim 1 or 2; (2) Pfu-mut-Cren7 engineered DNA polymerase, wherein the DNA polymerase is based on Pfu polymerase of the archaea B family, including at least one mutation of V93Q, E148K, G150A, S152A or N155A, and the C-terminus of the Pfu polymerase is fused with Cren7 DNA binding protein via a short linker peptide. (3) High GC PCR system, the components of which include: LiCl, MgCl2, dNTP, 7-deaza-dGTP, betaine, DMSO, propylene glycol, trehalose and nonionic surfactant.

4. The multiplex PCR detection kit according to claim 3, characterized in that, The components of the high-GC PCR system include: 20-120 mM LiCl, 1.5-3.5 mM MgCl2, 8-12 mM dNTP, 100-300 μM 7-deaza-dGTP, 0.8-1.5 M betaine, 3%-8% (v / v) DMSO, 1%-3% (v / v) propylene glycol, 0.2-0.4 M trehalose, and 0.005%-0.02% (v / v) nonionic surfactant.

5. The multiplex PCR detection kit according to claim 4, characterized in that, The components of the high GC PCR system also include: 5~15mM KCl, 5~15mM (NH4)2SO4, and 10~30mM Tris-HCl.

6. The multiplex PCR detection kit according to claim 3, characterized in that, The amino acid sequence of the Pfu-mut-Cren7 engineered DNA polymerase is shown in SEQ ID NO:

8.

7. A multiplex PCR detection method, characterized in that, The method is for non-disease treatment and diagnosis purposes. The method uses the reagents and the sample template from the multiplex PCR detection kit according to any one of claims 3-6, mixes them, and then performs a PCR reaction. The reaction procedure includes: Pre-denaturation at 98℃ for 1-5 minutes; Denaturation at 98℃ for 15-30 seconds, annealing at 58-65℃ for 30 seconds, extension at 75-78℃ for 1-3 minutes, cycle 28-35 times; Incubate at 75~78℃ for 3~6 min to obtain the amplified product, then store at 4℃.

8. The multiplex PCR detection method according to claim 7, characterized in that, The amplification products were detected by capillary electrophoresis or agarose gel electrophoresis. The target bands or peaks of FXS, NIID, and OPDM2 corresponded to the CGG repeat amplification fragments in their respective non-coding regions.

9. The use of the multiplex PCR primer set as described in claim 1 or 2, or the multiplex PCR detection kit as described in any one of claims 3 to 6, in the preparation of reagents for the detection of CGG repeat amplification disease.

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