Double-probe and primer composition for detecting HLA-B*58: 01 allele

Abstract

The invention discloses a double-probe and primer composition for detecting HLA-B * 58: 01 alleles, and relates to the technical field of biotechnology and genetic diagnose.The double-probe and primer composition detects the HLA-B * 58: 01 alleles with non-diagnosis and non-treatment as the purpose through the following steps that template DNA is extracted from anticoagulation; performing real-time quantitative qPCR (quantitative polymerase chain reaction) amplification by taking the DNA extracted in the step 1 as a sample DNA; during qPCR amplification, FAM, HEX and CY5 three-channel fluorescence signals are respectively collected in real time, and HLA-B * 58: 01 alleles of a sample to be detected are analyzed; compared with the existing single-probe detection, the double-probe and primer composition for detecting the HLA-B * 58: 01 allele has the advantage that the detection accuracy and reliability can be remarkably improved by simultaneously using two specific probes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and gene diagnosis technology, in particular to a double probe and primer combination for detecting HLA-B*58:01 allele. Background Art

[0002] Gout is an acute and chronic inflammation and tissue damage caused by the deposition of monosodium urate in the bones, joints, kidneys and subcutaneous tissues. It is directly related to hyperuricemia caused by purine metabolism disorders and / or reduced uric acid excretion, and belongs to the category of metabolic rheumatic diseases. Allopurinol is the most commonly used drug for the treatment of gout and is recommended as a first-line drug in various domestic and international guidelines. The drug is inexpensive and long-term use will not increase the economic burden. However, some patients taking allopurinol will experience adverse reactions such as rash, fever, abnormal liver function, and even more serious symptoms such as Stevens-Johnson syndrome and toxic epidermal necrolysis. The occurrence of these adverse reactions is closely related to the presence of the HLA-B*58:01 allele.

[0003] HLA-B*58:01 is an allele of the human leukocyte antigen (HLA) that is associated with a higher incidence of gout in certain populations. Specifically, individuals carrying the HLA-B*58:01 allele have a significantly increased risk of gout. The proportion of the HLA-B*58:01 allele in the Chinese population is approximately 12%. This association is particularly evident in Asian populations, especially in studies in countries such as China, South Korea, and Thailand. In addition, carriers of the HLA-B*58:01 allele may experience severe allergic reactions when taking allopurinol, which also suggests the complex relationship between this gene and gout treatment.

[0004] Given the association between the HLA-B*58:01 allele and gout, doctors may need to adopt a more cautious strategy when treating gout for individuals who carry this gene. This includes genetic testing before using drugs such as allopurinol to avoid unnecessary adverse drug reactions, and may need to explore other safer and more effective treatment options. At the same time, this also provides new ideas for the prevention and personalized treatment of gout, that is, to identify high-risk groups through genetic testing and take corresponding preventive measures.

[0005] The HLA-B*58:01 gene is similar to multiple allele sequences such as HLA-B*15:02, HLA-B*15:25, HLA-B*57:01, HLA-B*15:17 and HLA-B*18:02. Traditional detection methods are insufficient in specificity and accuracy, which affects the accuracy of subsequent medication. Summary of the invention

[0006] To solve the above problems, the purpose of the present invention is to provide a dual probe and primer combination for detecting HLA-B*58:01 alleles to improve the specificity and accuracy of detecting HLA-B*58:01 alleles. This method can effectively avoid serious adverse reactions in gout patients during drug treatment and improve the safety and effectiveness of treatment.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A dual probe and primer combination for detecting HLA-B*58:01 allele, wherein the dual probe and primer are designed according to the HLA-B*58:01 allele-specific site, and the sequence is as follows:

[0009] First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X;

[0010] Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X;

[0011] Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′;

[0012] Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′.

[0013] Preferably, the dual probe and primer combination for detecting the HLA-B*58:01 allele further comprises an internal reference probe, an internal reference forward primer and an internal reference reverse primer, the sequences of which are as follows;

[0014] The internal reference probe:

[0015] β-Globin-probe: 5'CY5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X;

[0016] The internal reference forward primer (β-Globin-F): 5'-AGTCAGGGCAGAGCCATCTA-3';

[0017] The internal reference reverse primer (β-Globin-R): 5'-TTAGGGTTGCCCATAACAGC-3'.

[0018] Preferably, the dual probe and primer combination detects the HLA-B*58:01 allele for non-diagnostic and non-therapeutic purposes by the following steps:

[0019] Step 1, extracting template DNA from anticoagulated blood;

[0020] Step 2, using the DNA extracted in step 1 as sample DNA for qPCR amplification; preparing an internal reference probe and an internal reference primer combination during amplification;

[0021] The dual probe, probe primer, internal reference probe and internal reference primer compositions in qPCR amplification are as follows:

[0022] First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X;

[0023] Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X;

[0024] Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′;

[0025] Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′;

[0026] Step 3: During the qPCR amplification process, the three-channel fluorescence signals of FAM, HEX and CY5 are collected in real time to analyze the HLA-B*58:01 allele and the internal reference gene of the sample to be tested.

[0027] Further preferably, in step 2,

[0028] The internal reference probe:

[0029] β-Globin-probe: 5'cy5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X;

[0030] The internal reference forward primer: 5'-AGTCAGGGCAGAGCCATCTA-3';

[0031] The internal reference reverse primer: 5'-TTAGGGTTGCCCATAACAGC-3'.

[0032] Preferably, the qPCR amplification reaction system in step 2 includes:

[0033]

[0034] Preferably, the qPCR amplification reaction conditions in step 2 are: pre-denaturation: 94°C, 3 min; denaturation: 94°C, 5 s, 60°C, 30 s, 40 cycles.

[0035] The present invention also includes preparing the above-mentioned double probe and primer combination into a kit for detecting HLA-B*58:01 allele.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] Compared with the existing single probe detection, the dual probe and primer combination for detecting HLA-B*58:01 allele of the present invention can significantly improve the accuracy and reliability of detection by using two specific probes at the same time. This method can not only reduce the false alarm rate and missed detection rate, but also more accurately identify the target gene, thereby improving the specificity and accuracy of HLA-B*58:01 allele detection.

[0038] The double probe and primer combination for detecting HLA-B*58:01 allele of the present invention places the internal reference gene in the same tube for reaction, which not only reduces the operation steps, but also avoids the influence of the difference in reaction efficiency between different tubes on the experimental results, and further improves the accuracy and repeatability of the experiment. In addition, the double probe and primer combination of the present invention are reasonably designed, have high specificity and sensitivity, can accurately identify HLA-B*58:01 allele, and reduce the incidence of false positive and false negative. Therefore, the double probe and primer combination of the present invention have a wide range of application prospects in the detection of HLA-B*58:01 allele, and are particularly valuable in the individualized treatment and prevention of gout patients. At the same time, the kit provided by the present invention is easy to prepare, simple to operate, and easy to promote and apply. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Comparison of the sequences of HLA-B*58:01 allele with HLA-B*15:02, HLA-B*15:25 and other alleles, and recognition sites of the forward primer, reverse primer and probe of the present invention;

[0040] Figure 2 This is a schematic diagram of the results when the fluorescence signals of the three channels of FAM, HEX and CY5 are all amplified;

[0041] Figure 3 This is a schematic diagram of the results when a single-channel fluorescence signal is amplified;

[0042] Figure 4 This is a schematic diagram of the results of channel-free fluorescence signal amplification. DETAILED DESCRIPTION

[0043] The object of the present invention is to provide a dual probe and primer combination for detecting HLA-B*58:01 allele.

[0044] like Figure 1As shown, the HLA-B*58:01 gene is similar to multiple allele sequences such as HLA-B*15:02, HLA-B*15:25, HLA-B*57:01, HLA-B*15:17 and HLA-B*18:02. Therefore, when a single probe is used to detect the HLA-B*58:01 allele in the prior art, false positive results are likely to occur. The present invention designs two specific MGB probes for this allele, which respectively identify different sequence regions of the HLA-B*58:01 gene, thereby increasing the specificity of the detection.

[0045] A dual probe and primer combination for detecting HLA-B*58:01 allele, wherein the dual probe and primer are designed according to the HLA-B*58:01 allele-specific site, and the sequence is as follows:

[0046] First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X;

[0047] Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X;

[0048] Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′;

[0049] Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′.

[0050] When performing the detection, the existing internal reference probe, internal reference forward primer and internal reference reverse primer can be used to detect the HLA-B*58:01 allele.

[0051] The addition of the internal reference probe is to monitor the extraction quality, amplification efficiency and stability of the reaction system of the sample DNA during the qPCR amplification process. The internal reference probe usually selects a gene sequence that is stably expressed in various tissues and organs, such as the β-Globin gene, whose expression level is not affected by the disease state or therapeutic intervention. In the detection system of the present invention, the β-Globin internal reference probe and the HLA-B*58:01 allele probe are amplified in the same tube, and the fluorescence signal of the CY5 channel is collected in real time to reflect the amplification efficiency of the sample DNA. When the sample DNA quality is good and the amplification efficiency is stable, the fluorescence signal of the internal reference probe will show a stable growth trend. If the sample DNA quality is poor or the amplification efficiency is inhibited, the fluorescence signal of the internal reference probe will be abnormal, thereby prompting the experimenter to optimize the experimental conditions or re-extract the sample DNA. This internal reference monitoring mechanism helps to improve the accuracy and reliability of the test and ensure the accuracy of the experimental results. This internal reference gene can be replaced by other genes such as GAPDH, β-actin, etc., but it is not as stable as the β-Globin internal reference probe data preferred by the present invention.

[0052] Preferably, it also includes an internal reference probe, an internal reference forward primer and an internal reference reverse primer, the sequences of which are as follows:

[0053] The internal reference probe:

[0054] β-Globin-probe: 5'CY5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X;

[0055] The internal reference forward primer: 5'-AGTCAGGGCAGAGCCATCTA-3';

[0056] The internal reference reverse primer: 5'-TTAGGGTTGCCCATAACAGC-3'.

[0057] Preferably, the dual probe and primer combination detects the HLA-B*58:01 allele for non-diagnostic and non-therapeutic purposes by the following steps:

[0058] Step 1, extracting template DNA from anticoagulated blood;

[0059] Step 2, using the DNA extracted in step 1 as sample DNA for qPCR amplification;

[0060] The dual probe, probe primer, internal reference probe and internal reference primer compositions in qPCR amplification are as follows:

[0061] First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X;

[0062] Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X;

[0063] Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′;

[0064] Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′;

[0065] Step 3: During the qPCR amplification process, the three-channel fluorescence signals of FAM, HEX and CY5 are collected in real time to analyze the HLA-B*58:01 allele and the internal reference gene of the sample to be tested.

[0066] Further preferably, in step 2,

[0067] The internal reference probe:

[0068] β-Globin-probe: 5'cy5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X;

[0069] The internal reference forward primer: 5'-AGTCAGGGCAGAGCCATCTA-3';

[0070] The internal reference reverse primer: 5'-TTAGGGTTGCCCATAACAGC-3'.

[0071] Preferably, the qPCR amplification reaction system in step 2 includes:

[0072]

[0073]

[0074] Preferably, the qPCR amplification reaction conditions in step 2 are: pre-denaturation: 94°C, 3 min; denaturation: 94°C, 5 s, 60°C, 30 s, 40 cycles.

[0075] The present invention also includes preparing the above-mentioned double probe and primer combination into a kit for detecting HLA-B*58:01 allele.

[0076] The present invention is further described below in conjunction with specific embodiments.

[0077] Example 1

[0078] The dual probe and primer combination is used to detect HLA-B*58:01 allele for non-diagnostic and non-therapeutic purposes, comprising the following steps:

[0079] Step 1, extracting template DNA from anticoagulated blood;

[0080] DNA was extracted according to the instructions of a commercial blood genomic DNA extraction kit (Tiangen Biochemical Technology, DP348) as follows:

[0081] Take 200μl whole blood sample (EDTA or heparin anticoagulation) to extract sample DNA. Add 200μl buffer GB and 20μl proteinase K premixed solution to 200μl blood, mix thoroughly by inversion, place at 56℃ for 10min, invert several times during the mixture, and the solution should become clear. After placing at room temperature for 2-5min, add 350μl buffer BD and mix thoroughly by inversion. Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CG2 (adsorption column CG2 is placed in a collection tube), centrifuge at 12000rpm (~13400×g) for 30s, discard the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube. Add 500μl buffer GDB to the adsorption column CG2, centrifuge at 12000rpm (~13400×g) for 30s, discard the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube. Add 600μl of rinsing solution PWB to the adsorbed CG2, centrifuge at 12000rpm (~13400×g) for 30s, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube. Repeat the rinse once. Leave the adsorption column CG2 at room temperature for 2 minutes to thoroughly dry the residual rinsing solution in the adsorption material. Transfer the adsorption column CG2 to a 1.5ml centrifuge tube, add 50μl of elution buffer TB to the middle of the adsorption membrane, leave it at room temperature for 2 minutes, centrifuge at 12000rpm (~13400×g) for 2 minutes, and collect the DNA in a centrifuge tube. The concentration is 30-50ng / μl.

[0082] Step 2, using the DNA extracted in step 1 as sample DNA for qPCR amplification;

[0083] The dual probe, probe primer, internal reference probe and internal reference primer compositions in qPCR amplification are as follows:

[0084] First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X;

[0085] Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X;

[0086] Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′;

[0087] Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′;

[0088] Internal reference probe:

[0089] β-Globin-probe: 5'CY5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X;

[0090] Internal reference forward primer: 5′-AGTCAGGGCAGAGCCATCTA-3′;

[0091] Internal control reverse primer: 5′-TTAGGGTTGCCCATAACAGC-3′;

[0092] Preparation of primers and probes: Find the sequence of this allele in NCBI and use PRIMER SOFTWARE V3.0.1 was used to design probes and primers. This software can be designed according to TAQMAN or TAQMAN MGB detection methods, and can be adjusted manually locally to further optimize the amplification efficiency of probes and primers.

[0093] The designed probe primer sequences were synthesized by Shanghai Bioengineering.

[0094] Prepare a 100 μM stock solution with ddH2O according to the primer design report. Further dilute the primers and probes to 10 μM. Dissolve and mix, centrifuge briefly at 100×g for 10 seconds, and set aside.

[0095] The qPCR amplification reaction system includes:

[0096]

[0097]

[0098] The raw materials of the above qPCR amplification reaction system were mixed and briefly centrifuged at 100×g for 10s.

[0099] Select Dual Color Hydrolysis probe / UPL probe on the light cycler 480 and run qPCR according to the following program.

[0100] qPCR amplification reaction conditions: pre-denaturation: 94°C, 3 min; denaturation: 94°C, 5 s, 60°C, 30 s, 40 cycles.

[0101] Step 3: During the qPCR amplification process, the three-channel fluorescence signals of FAM, HEX and CY5 were collected respectively to analyze the HLA-B*58:01 allele and the internal reference gene of the sample to be tested; the fluorescence signal was collected after each cycle during the amplification process until the end of 40 cycles.

[0102] When the fluorescence signals of the three channels of FAM, HEX and CY5 are all amplified, the results are as follows: Figure 2 As shown; the single channel amplification results are shown Figure 3 As shown, the results of no channel amplification are as follows Figure 4 As shown. Figure 2 to Figure 4 In the figure, the ordinate is the fluorescence intensity and the abscissa is the number of cycles. When the probe is negative, the fluorescence value is very low regardless of the number of cycles.

[0103] exist Figure 2 As the number of cycles increases, the fluorescence intensity on the ordinate increases significantly, indicating that positive peaks appear in all three channels of FAM, HEX, and CY5. Figure 3 Only the internal reference probe has a peak, and the other two have no peaks; Figure 4 The fluorescence in the middle fluctuates around the very low background fluorescence intensity, and the fluorescence has not increased. None of the three probes have peaks. Figure 3 and Figure 4 All were judged as negative.

[0104] From 300 patient specimens, 20 pyrophosphate sequencing positive specimens and 40 negative specimens were selected and tested using the method of Example 1. The results are shown in Table 1.

[0105] Table 1 Detection results using the method of Example 1

[0106]

[0107]

[0108] It can be seen from the results in Table 1 that the double probe and primer combination of the present invention detects HLA-B*58:01 allele, and compared with the gold standard method, the negative and positive coincidence rates are both 100%.

[0109] 24 patient specimens were tested using the gold standard method, a commercial single probe method, and the dual probe and primer combination detection method of the present invention. The test results are shown in Table 2.

[0110] Table 2 Test results

[0111] Pyrosequencing (gold standard) Single probe Dual probe and primer combination method of the present invention Positive 17 24 17 False Positive Rate -- 29％ 0

[0112] It can be seen from the results in Table 2 that the false positive rate reaches 29% when the existing single probe is used for detection, while the false positive rate is 0% when the dual probe and primer combination method of the present invention is used for detection. It can be seen that the dual probe and primer combination method of the present invention greatly reduces the occurrence of false positive rate.

Claims

1. A dual probe and primer combination for detecting HLA-B*58:01 allele, characterized in that: The dual probe and primers were designed based on the HLA-B*58:01 allele-specific site, and the sequences are as follows: First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X; Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X; Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′; Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′.

2. The dual probe and primer combination for detecting HLA-B*58:01 allele according to claim 1, characterized in that: Also included are an internal reference probe, an internal reference forward primer, and an internal reference reverse primer, the sequences of which are as follows; The internal reference probe: β-Globin-probe: 5'CY5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X; The internal reference forward primer: 5'-AGTCAGGGCAGAGCCATCTA-3'; The internal reference reverse primer: 5'-TTAGGGTTGCCCATAACAGC-3'.

3. The dual probe and primer combination according to claim 1, characterized in that: The dual probe and primer combination detects the HLA-B*58:01 allele for non-diagnostic and non-therapeutic purposes by the following steps: Step 1, extracting template DNA from anticoagulated blood; Step 2, using the DNA extracted in step 1 as sample DNA for qPCR amplification; preparing an internal reference probe and an internal reference primer combination during amplification; The dual probes and probe primers used in qPCR amplification are as follows: First probe: 5′6-FAM-ACCGAGAGAACCTGCGGATCGCGCTCC-3′BHQ-X; Second probe: 5′HEX-TCCGAGATCCGCCTCCCTGAGGCC-3′BHQ-X; Forward primer: 5′-GGGCCGGAGTATTGGGATG-3′; Reverse primer: 5′-TGAAACCGGGTAAACGCG-3′; Step 3: During qPCR amplification, the three-channel fluorescence signals of FAM, HEX and CY5 are collected respectively to analyze the HLA-B*58:01 allele and the internal reference gene of the sample to be tested.

4. The dual probe and primer combination according to claim 3, characterized in that: The internal reference probe: β-Globin-probe: 5'CY5-AGTCTGCCGTTACTGCCCTGTGG-3'BHQ-X; The internal reference forward primer: 5'-AGTCAGGGCAGAGCCATCTA-3'; The internal reference reverse primer: 5'-TTAGGGTTGCCCATAACAGC-3'.

5. The dual probe and primer combination according to claim 3 or 4, characterized in that: The qPCR amplification reaction system in step 2 includes:

6. The dual probe and primer combination according to claim 3, characterized in that: The qPCR amplification reaction conditions in step 2 were as follows: pre-denaturation: 94°C, 3 min; denaturation: 94°C, 5 s, 60°C, 30 s, 40 cycles.

7. The dual probe and primer combination according to claim 1, characterized in that: A kit for detecting the HLA-B*58:01 allele is prepared.

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