A LHON detection kit

Abstract

The present invention provides a Leber Hereditary Optic Neuropathy (LHON) detection kit, which detects m.11778G>A of MT-ND4 gene, m.14484T>C of MT-ND6 gene and m.3460G>A of MT-ND1 gene for diagnosis of LHON by optimizing the primer and probe combination of LHON-related mutation sites, and optimizes the qPCR reaction system. The kit provided by the present invention has high detection sensitivity, good stability, strong practicality, is less affected by interference factors, and has good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR kit for LHON detection. Background Art

[0002] Leber Hereditary Optic Neuropathy (LHON) is a maternally inherited disease that usually presents as painless acute vision loss in both eyes. The disease is associated with mitochondrial DNA mutations. Currently disclosed pathogenic mutations include: m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, and m.3460G>A in the MT-ND1 gene.

[0003] Currently, PCR is mostly used to detect mutations in the detection of LHON.

[0004] The Chinese patent application number CN201611076186.4 discloses a detection primer, kit and detection method for mitochondrial DNA gene mutations in Leber hereditary optic neuropathy. Based on the principle of Sanger sequencing, the four most common pathogenic mutations of LHON-mtDNA are detected simultaneously, solving the problems of low sensitivity and insufficient specificity, easy contamination and high cost of existing methods for detecting LHON mitochondrial DNA mutations, providing an accurate genetic diagnosis method for LHON, which is of great significance for genetic counseling and gene therapy of the disease.

[0005] The Chinese patent application number CN201410128241.4 discloses: a Leber hereditary optic neuropathy in vitro diagnostic kit, including 12 primers. The kit can achieve the prevention and early diagnosis of LHON, identify high-risk groups, and improve the diagnosis rate of LHON.

[0006] However, the sensitivity of the detection methods in the existing technology is relatively low and there is room for further improvement. Optimizing primers and amplification systems is a good direction.

[0007] The above detection methods all adopt the PCR detection method, that is, the fragment of the corresponding mutation site is amplified by the polymerase chain reaction for detection. The biggest feature of PCR is that it can greatly increase the trace amount of DNA, but it is still affected by the concentration and quality of the template. PCR uses the DNA template to denature into a single strand when treated at high temperature in vitro. When treated at low temperature, the primer and the single-stranded template are combined according to the principle of base complementary pairing, and then the temperature is adjusted to the optimal reaction temperature of the DNA polymerase. The DNA polymerase synthesizes a complementary chain along the direction from phosphate to pentose (5'-3'). In this reaction process, it is easily affected by temperature changes. The PCR instrument in the prior art has solved this problem well, but in the PCR reaction process, the primer probe sequence designed for different amplification target fragments, and the addition of various raw materials or enhancers in the amplification system, but there is no fixed form. The PCR kit for detecting LHON in the prior art needs to improve the sensitivity, and research in this direction can be focused on to better apply it to clinical detection. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a LHON PCR detection kit, adjusts the primer probe combination, and optimizes the qPCR reaction system, aiming to improve the detection effect of LHON, reduce the detection limit, and improve the detection accuracy and anti-interference ability.

[0009] In one aspect, the present invention provides a primer-probe combination for detecting LHON.

[0010] The primer-probe combination is used to detect the m.3460G>A mutation of the MT-ND4 gene, the m.11778G>A mutation of the MT-ND4 gene and the m.14484T>C mutation of the MT-ND4 gene.

[0011] The m.3460G>A detection primer probe comprises:

[0012] Forward primer F1: (SEQ ID NO.1);

[0013] Reverse primer R1: (SEQ ID NO.2);

[0014] Probe1-G3460: (SEQ ID NO. 7), 5'-end modified with VIC, 3'-end modified with MGB;

[0015] Probe2-A3460: (SEQ ID NO. 8), the 5' end is modified with FAM, and the 3' end is modified with MGB.

[0016] The m.11778G>A detection primer probe comprises:

[0017] Forward primer F2: (SEQ ID NO.3);

[0018] Reverse primer R2: (SEQ ID NO.4);

[0019] Probe3-G11778: (SEQ ID NO. 9), modified with VIC at the 5' end and MGB at the 3' end;

[0020] Probe4-A11778: (SEQ ID NO. 10), modified with FAM at the 5' end and MGB at the 3' end;

[0021] The m.14484T>C detection primer probe comprises:

[0022] Forward primer F3: (SEQ ID NO.5);

[0023] Reverse primer R3: (SEQ ID NO.6);

[0024] Probe5-T14484: (SEQ ID NO. 11), modified with VIC at the 5' end and MGB at the 3' end;

[0025] Probe6-C14484: (SEQ ID NO. 12), 5'-end modified with FAM, 3'-end modified with MGB;

[0026] Preferably, in the primer-probe mixture, the concentrations of F1 / R1, F2 / R2 and F3 / R3 are all 0.4 μM.

[0027] Preferably, in the primer-probe mixture, the concentrations of Probe1-G3460, Probe2-A3460, Probe3-G11778, Probe4-A11778, Probe5-T14484 and Probe6-C14484 are all 0.2 μM.

[0028] In yet another aspect, the present invention provides use of the aforementioned mixture in preparing a kit for detecting LHON.

[0029] The kit is a qPCR detection kit.

[0030] The kit includes the aforementioned primer-probe mixture.

[0031] The kit also includes other reagents for qPCR reaction.

[0032] The other reagents used for qPCR reaction include but are not limited to: DNA polymerase, dNTPs, and buffer.

[0033] In yet another aspect, the present invention provides a kit for detecting LHON.

[0034] The kit includes the mixture of the primers and probes mentioned above, and the kit is a qPCR detection kit.

[0035] The kit also includes betaine and metal ions.

[0036] Preferably, the betaine is used at a concentration of 1-2M, more preferably 1.5-2M, and further preferably 1.5M.

[0037] The metal ions include magnesium ions, potassium ions and aluminum ions.

[0038] Preferably, the molar ratio of magnesium ion:potassium ion:aluminum ion is 1-3:3-10:2-5, and more preferably 1:4:2.

[0039] The total concentration of the metal ions used is 50-100 mM, preferably 70-100 mM, and more preferably 70 mM.

[0040] The source of the metal ions can be metal salts, such as chlorides, sulfates, and the like.

[0041] Preferably, the metal ions are derived from their chlorides.

[0042] The kit also includes other reagents for qPCR reaction.

[0043] The other reagents used for qPCR reaction include but are not limited to: DNA polymerase, dNTPs, and buffer.

[0044] Beneficial effects of the present invention:

[0045] The present invention optimizes the primer and probe combination of LHON-related mutation sites, diagnoses LHON by detecting m.11778G>A of the MT-ND4 gene, m.14484T>C of the MT-ND6 gene, and m.3460G>A of the MT-ND1 gene, and optimizes the qPCR reaction system. The kit provided by the present invention has high detection sensitivity, good stability, strong practicality, and is less affected by interference factors, and has a good clinical application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the detection result of the m.3460G>A site.

[0047] Figure 2 This is the detection result of the m.11778G>A site.

[0048] Figure 3 This is the detection result of the m.14484T>C site.

[0049] Figure 4 The test results of the test kit are stored for 24 weeks. DETAILED DESCRIPTION

[0050] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0051] Example 1

[0052] A LHON qPCR detection kit, comprising:

[0053] (1) Primers and probes were commissioned to be synthesized by Shanghai Bioengineering, as follows:

[0054] F1: SEQ ID NO.1;

[0055] R1: SEQ ID NO.2;

[0056] Probe1-G3460: SEQ ID NO.7, 5'-end modified with VIC, 3'-end modified with MGB;

[0057] Probe2-A3460: SEQ ID NO. 8, with FAM modification at the 5' end and MGB modification at the 3' end.

[0058] F2: SEQ ID NO.3;

[0059] R2: SEQ ID NO.4;

[0060] Probe3-G11778: SEQ ID NO.9, 5'-end modified with VIC, 3'-end modified with MGB;

[0061] Probe4-A11778: SEQ ID NO.10, 5'-end modified with FAM, 3'-end modified with MGB;

[0062] F3: SEQ ID NO.5;

[0063] R3: SEQ ID NO.6;

[0064] Probe5-T14484: SEQ ID NO. 11, 5'-end modified with VIC, 3'-end modified with MGB;

[0065] Probe6-C14484: SEQ ID NO. 12, 5'-end modified with FAM, 3'-end modified with MGB;

[0066] The primers were used at a concentration of 0.4 μM, and the probes were used at a concentration of 0.2 μM.

[0067] (2) Magnesium chloride, potassium chloride, aluminum chloride;

[0068] (3) Betaine;

[0069] (4) Positive control: a mixture of 3460-site mutant, 11778-site mutant, and 14484-site mutant recombinant plasmids (concentration 1×10 5 copies / μL, commissioned by Shanghai Bioengineering Group for synthesis);

[0070] (5) Negative control: a mixture of wild-type recombinant plasmids at site 3460, site 11778, and site 14484 (concentration 1×10 5 copies / μL, commissioned by Shanghai Bioengineering Group for synthesis);

[0071] (6) dNTPs;

[0072] (7) Hot start Taq enzyme: Yisheng, 10717ES72;

[0073] (8)10×Buffer.

[0074] The qPCR amplification system of this kit is:

[0075] Reagents Dosage 10×Buffer 2μL Magnesium chloride (1M) 0.2μL Potassium chloride (1M) 0.8μL Aluminum chloride (1M) 0.4μL Betaine(30M) 1μL dNTPs (2.5 mM) 1μL Taq enzyme (5U / μL) 0.5μL template 2μL 3460 / 11778 / 14484 locus primer probe mixture 2μL water Make up to 20 μL

[0076] The reaction conditions are:

[0077]

[0078] Example 2

[0079] The difference from Example 1 is that in the qPCR amplification system: the amount of magnesium chloride added is 0.3 μL, the amount of potassium chloride added is 1 μL, and the amount of aluminum chloride added is 0.5 μL.

[0080] Example 3

[0081] The difference from Example 1 is that in the qPCR amplification system: the amount of magnesium chloride added is 0.4 μL, the amount of potassium chloride added is 0.6 μL, and the amount of aluminum chloride added is 0.4 μL.

[0082] Experimental Example 1 Actual Sample Testing

[0083] The kits in Examples 1-3 were tested.

[0084] Sample extraction: Use a commercially available nucleic acid extraction kit (Jinmaig, NA006-1) to extract oral swab samples. The extracted DNA should be tested immediately, otherwise it should be stored at -20°C. The storage time should not exceed 6 months and avoid repeated freezing and thawing. The concentration of extracted DNA should not be less than 1 ng / μL, and A260 / A280 should be between 1.8-2.0.

[0085] The positive and negative quality control products of the kit do not need to be extracted and can be used directly.

[0086] PCR amplification and sequencing: The extracted DNA was amplified using the primers and probes in Examples 1-3, respectively. After the amplified products were electrophoresed to check for correct sizes, they were purified and sent for Sanger sequencing to verify the results by comparing the sequences.

[0087] Oral swabs were collected from 17 LHON patients (5 patients with m.3460G>A heterozygous mutation, 6 patients with m.11778G>A heterozygous mutation, and 6 patients with m.14484T>C heterozygous mutation) and 30 healthy volunteers, and the kit of Example 1 was used for detection. The results showed that the negative coincidence rate of the kits provided in Examples 1-3 for detecting the m.3460G>A site through oral swabs was 100%, and the positive coincidence rate was 100%; the negative coincidence rate of detecting the m.11778G>A site was 100%, and the positive coincidence rate was 100%; the negative coincidence rate of detecting the m.14484T>C site was 100%, and the positive coincidence rate was 100%.

[0088] Due to the limited number of patients, in order to further evaluate the accuracy of the kit, oral swabs were collected from another 100 healthy volunteers for testing. The results showed that the negative coincidence rate of the kit provided in Examples 1-3 for detecting m.3460G>A, m.11778G>A and m.14484T>C sites was 100%, and the misdiagnosis rate was low.

[0089] Experimental Example 2 Quality Control Product Testing

[0090] Referring to the detection method of Experimental Example 1, the artificially synthesized 3460, 11778, 14484 site wild-type plasmids, mutant plasmids and a 1:1 mixture of the two were detected.

[0091] The test results of the kit of Example 1 are shown in Figure 1-3 .

[0092] It can be seen that the effect of the kit of the present application is: the detection accuracy is high, and the wild type, homozygous mutant type and mixed mutant type of each site can be effectively distinguished.

[0093] Experimental Example 3 Minimum Detection Limit of the Kit

[0094] In order to verify the detection limit of the kit of Examples 1-3, the positive control was diluted to different concentrations (1 coipes / μL, 10 coipes / μL, 100 coipes / μL, 1000 coipes / μL, 10000 coipes / μL), and the detection was carried out according to the method of Experimental Example 1. The results showed that the kit of Example 1 can detect a positive control of 1 coipes / μL in the amplification system at the lowest, and the kit of Examples 2-3 can detect a positive control of 10 coipes / μL, and has a higher sensitivity.

[0095] Experimental Example 4 Storage stability of the kit

[0096] The test kit was subjected to a storage stability test at -20±5°C and a relative humidity of 40-60%. The test kit was placed for 2 weeks, 4 weeks, 8 weeks, 16 weeks, and 24 weeks, respectively. After completion, the positive quality control product was tested according to the method of Experimental Example 2. The results showed that the detection effect was consistent with that of the newly prepared test kit. Figure 4 This is the detection effect of the positive quality control product in Example 1 after being placed for 24 weeks.

[0097] Experimental Example 5 Anti-interference ability of the kit

[0098] The purpose of this example is to detect the anti-interference ability of the kits of Examples 1-3 to ethanol.

[0099] The specific detection method is: for the positive quality control product of 10 coipes / μL, ethanol is added thereto at a volume ratio of 1%, 3%, 5%, 8%, and 10%, respectively, and then the kits of Examples 1-3 are used for detection. The results show that the kit of Example 1 can still detect normally under the condition of adding 10% ethanol, Example 2 can still detect normally under the condition of 8% ethanol, and Example 3 can still detect normally under the condition of 8% ethanol.

[0100] Comparative Example

[0101] The comparative example 1 is set as follows:

[0102]

[0103]

[0104] The sensitivity and anti-interference ability of the comparative sample kit were tested, and the results were as follows:

[0105] Sensitivity Maximum tolerance concentration of ethanol Comparative Example 1 10coipes / μL 5％ Comparative Example 2 100coipes / μL 8％ Comparative Example 3 10coipes / μL 5％ Comparative Example 4 100coipes / μL 3％ Comparative Example 5 100coipes / μL 5％ Comparative Example 6 100coipes / μL 3％ Comparative Example 7 No amplification -

Claims

1. A LHON detection kit, characterized in that: A primer-probe combination for detecting LHON is included, wherein the primer-probe combination comprises: The primer sequences shown in SEQ ID NOs. 1-6, and the probe sequences shown in SEQ ID NOs. 7-12; The probe was modified with VIC or FAM at the 5' end and MGB at the 3' end; The primers were used at a concentration of 0.4 μM, and the probes were used at a concentration of 0.2 μM; The kit also includes betaine and metal ions; the concentration of betaine used is 1.5M; The metal ions include magnesium ions, potassium ions and aluminum ions, wherein the molar ratio of magnesium ions: potassium ions: aluminum ions is 1:4:2; The total concentration of the metal ions used is 70 mM; the metal ions are derived from chloride.

2. The kit according to claim 1, characterized in that The kit also includes one or more of DNA polymerase, dNTPs and buffer.

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