Detection primer and kit for rapidly distinguishing HLA-A*24: 02 / HLA-A*24: 353 alleles and application of detection primer and kit

Abstract

The invention provides a detection primer and a kit for rapidly distinguishing HLA-A * 24: 02 / HLA-A * 24: 353 alleles and application of the detection primer and the kit, and belongs to the technical field of biomedicine. The invention discloses a detection primer for distinguishing HLA-A * 24: 02 / HLA-A * 24: 353 alleles. The nucleotide sequence of the detection primer is shown as SEQ ID NO: 1-2. According to the invention, HLA-A * 24: 02 / HLA-A * 24: 353 alleles are distinguished by adopting a fluorescent quantitative PCR (Polymerase Chain Reaction) method, and the typing result of a sample can be directly judged according to an amplification curve and whether a Ct value exists or not. The kit provided by the invention overcomes the current situation that the HLA-A * 24: 02 / HLA-A * 24: 353 allele can be distinguished only through PCR amplification and nucleic acid electrophoresis in the existing SSP method, so that the detection is quick and convenient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a detection primer and a kit for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, and an application thereof. Background Art

[0002] Human Leukocyte Antigen (HLA) is an important antigen system in the human body, located on the broken arm of chromosome 6. It is mainly responsible for identifying and presenting foreign antigens, thereby initiating immune responses. It is currently the most complex genetic polymorphism system in humans. HLA is divided into class I and class II according to the structural characteristics of the encoded protein. HLA-A is a classic HLA class I locus with rich diversity and important clinical significance in solid organ transplantation or hematopoietic stem cell transplantation. HLA-A allele mismatch may lead to severe graft-versus-host disease or transplant rejection and other adverse reactions that affect the patient's prognosis and survival. Therefore, high-resolution typing of the HLA-A gene of donors and recipients before transplantation is an important means to reduce adverse reactions and improve treatment effects.

[0003] When using Sanger sequencing technology (SBT) or oligonucleotide probe hybridization (SSO) for HLA genotyping, when encountering the result of HLA-A*24:02:01G (including HLA-A*24:02 or HLA-A*24:353), since HLA-A*24:02 and HLA-A*24:353 only have one different base at base 1018 of exon 6, neither SBT nor SSO can accurately determine it, and it is necessary to distinguish it through sequence-specific primer amplification (SSP) and nucleic acid electrophoresis. Although the development and application of next-generation sequencing technology (NGS) has been able to solve this problem through full-length sequencing, this method is time-consuming and requires 48-72 hours to produce results. When encountering expedited samples in clinical practice, it is often required to issue a test report within 24 hours. In this case, SBT or SSO, which is shorter in time, is still required. However, these two technologies are currently unable to distinguish between A*24:02 / A*24:353, and SSP amplification and nucleic acid electrophoresis are needed to make the final judgment. Nucleic acid electrophoresis involves gel preparation, spotting, electrophoresis and gel imaging, and requires gel preparation tools, electrophoresis instruments, imagers and other equipment. The steps are cumbersome and the equipment requirements are high. It also introduces some unstable factors (including agarose concentration, fluorescent dye intensity, electrophoresis conditions, etc.), which may affect the result judgment. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a detection primer for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, which belongs to the primers for fluorescence quantitative PCR amplification. The HLA-A*24:02 and HLA-A*24:353 alleles are determined according to the amplification curve and the presence or absence of Ct value, so as to achieve the purpose of fast and convenient detection.

[0005] The present invention provides a detection primer for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0006] The present invention provides a detection kit for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles based on fluorescent quantitative PCR technology, comprising the detection primers.

[0007] Preferably, the detection kit further comprises SYBR Green Mix.

[0008] Preferably, a sample genomic DNA extraction reagent is also included.

[0009] The present invention provides the use of the detection primer in preparing a rapid HLA-A*24:02 / HLA-A*24:353 allele detection kit.

[0010] The present invention provides a method for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, comprising the following steps:

[0011] Using the genomic DNA of the sample to be tested as a template, the reaction system is configured using the detection primers to perform qPCR detection, and the typing result of the sample is determined based on the amplification curve of the sample to be tested and the presence or absence of a Ct value:

[0012] When the sample to be tested has an amplification curve and the Ct value is ≤40, the sample to be tested contains the HLA-A*24:353 allele;

[0013] When there is no amplification curve for the sample to be tested and the Ct value is greater than 40, the sample to be tested contains the HLA-A*24:02 allele.

[0014] Preferably, the reaction system is 10 μL, specifically including 5 μL of SYBR Green Mix, 0.5 μL of 20 μM upstream primer, 0.5 μL of 20 μM downstream primer, 2 μL of genomic DNA and 2 μL of ddH2O.

[0015] Preferably, the concentration of the genomic DNA is 25 to 200 ng / μL; the OD of the genomic DNA is 260 / OD 280 It is 1.65~1.8.

[0016] Preferably, the reaction procedure of the qPCR detection is 95°C for 2 min; 95°C for 15 s, 55°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 2 min.

[0017] Preferably, the sample to be tested includes at least one of the following: blood, tissue fluid and secretion fluid.

[0018] The detection primers for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles provided by the present invention include a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2. The present invention designs a set of specific detection primers based on the amplification primer design principle of fluorescent quantitative PCR technology, and the primers can determine HLA-A*24:02 / HLA-A*24:353 alleles according to the amplification curve and the presence or absence of Ct value. The technical solution of the present invention overcomes the current situation that the existing SSP method needs to be amplified by PCR and nucleic acid electrophoresis to distinguish HLA-A*24:02 / HLA-A*24:353 alleles, making the detection fast and convenient. The kit of the present invention is simple to use, has high accuracy, can be monitored in the process, and has fast results, and there is no electrophoresis process, which is conducive to clinical promotion and popularization.

[0019] The present invention provides a detection kit for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles based on fluorescent quantitative PCR technology, including the detection primers. The present invention uses fluorescent quantitative PCR technology to determine HLA-A*24:02 / HLA-A*24:353 alleles, and can directly judge the typing results of the sample according to the amplification curve and the presence or absence of Ct value. The kit of the present invention overcomes the current situation that the existing SSP method needs PCR amplification and nucleic acid electrophoresis to distinguish HLA-A*24:02 / HLA-A*24:353 alleles, making the detection fast and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the results of 31 samples with known genotypes in Example 2 tested using the kit of the present invention;

[0021] Figure 2 The amplification curves of 31 samples with known genotypes detected by the kit of the present invention in Example 2 are as follows;

[0022] Figure 3This is a graph showing the test results of using the kit of the present invention to distinguish HLA-A*24:02 / HLA-A*24:353 alleles in a sample containing HLA-A*24:02:01G detected by SBT in Example 3;

[0023] Figure 4 This is an amplification curve diagram of the sample containing HLA-A*24:02:01G detected by SBT in Example 3 using the kit of the present invention to distinguish the HLA-A*24:02 / HLA-A*24:353 alleles;

[0024] Figure 5 The electrophoresis diagram of the test of the sample with known genotype by using the existing commercial kit in Comparative Example 1;

[0025] Figure 6 This is a graph showing the test results of the control primers for the samples with known genotypes in Comparative Example 2;

[0026] Figure 7 The amplification curve of the sample with known genotype using the control primer in Comparative Example 2;

[0027] Figure 8 This is a graph showing the test results for samples with known genotypes in Comparative Example 3;

[0028] Fig. 9 This is the amplification curve diagram of the sample with known genotype in Comparative Example 3. DETAILED DESCRIPTION

[0029] The present invention provides a detection primer for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, comprising a forward primer having a nucleotide sequence as shown in SEQ ID NO:1 (TTCTTCCCACAGATAGAG) and a reverse primer having a nucleotide sequence as shown in SEQ ID NO:2 (CTGGTCAGATCCCACAGAA).

[0030] The present invention has no special restrictions on the source of the detection primer, and the gene synthesis method known in the art can be used. In the embodiment of the present invention, the detection primer is commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0031] The present invention provides a detection kit for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles based on fluorescent quantitative PCR technology, comprising the detection primers.

[0032] In the present invention, the detection kit preferably further comprises SYBR Green Mix. The SYBR Green Mix preferably comprises SYBR Green dye, DNA polymerase, buffer, dNTPs (deoxynucleotide triphosphates), Mg 2+ (Magnesium ion). In the embodiment of the present invention, the SYBR Green Mix is ​​purchased from Thermo Fisher Scientific.

[0033] In the present invention, the detection kit preferably also includes a sample genomic DNA extraction reagent. The present invention has no special restrictions on the type of sample genomic DNA extraction reagent, and a sample genomic DNA extraction reagent known in the art can be used. In an embodiment of the present invention, the sample genomic DNA extraction reagent is purchased from Ruibao Biomedical Technology Co., Ltd.

[0034] The present invention provides the use of the detection primer in preparing a detection kit for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles.

[0035] The present invention provides a method for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, comprising the following steps:

[0036] Using the genomic DNA of the sample to be tested as a template, the reaction system is configured using the detection primers to perform qPCR detection, and the typing result of the sample is determined based on the amplification curve of the sample to be tested and the presence or absence of a Ct value:

[0037] When the sample to be tested has an amplification curve and the Ct value is ≤40, the sample to be tested contains the HLA-A*24:353 allele;

[0038] When there is no amplification curve for the sample to be tested and the Ct value is greater than 40, the sample to be tested contains the HLA-A*24:02 allele.

[0039] In the present invention, the reaction system is preferably 10 μL, specifically including 5 μL of SYBR Green Mix, 0.5 μL of 20 μM upstream primer, 0.5 μL of 20 μM downstream primer, 2 μL of genomic DNA and 2 μL of ddH2O. The concentration of the genomic DNA is preferably 25 to 200 ng / μL, can be 50 to 150 ng / μL, or can be 100 ng / μL. The OD of the genomic DNA is 260 / OD 280is 1.65 to 1.8, and can be 1.70 to 1.75. The present invention has no special restrictions on the extraction method of genomic DNA of the sample to be tested, and an extraction method known in the art can be used. The reaction procedure of the qPCR detection is preferably 95°C 2min; 95°C 15s, 55°C 15s, 72°C 45s, 40 cycles; 72°C 2min.

[0040] In the present invention, the sample to be tested preferably includes at least one of the following: blood, tissue fluid and secretion fluid.

[0041] In the embodiment of the present invention, the above method is used to detect samples with known genotypes, and the results show that the consistency reaches 100%. Compared with other conventionally designed detection primers, the detection primers provided by the present invention can accurately distinguish HLA-A*24:02 and HLA-A*24:353 alleles, avoiding the problem of false negative or false positive test results. At the same time, compared with the method of commercial kit PCR detection combined with electrophoresis detection, the detection method of the present invention is simple, highly accurate, the process can be monitored, and the results are fast, which is conducive to clinical promotion and popularization.

[0042] The following is a detailed description of the detection primers, kit and application of the present invention for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A detection kit for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, comprising the following reagents:

[0045] 1. Primer-F: 5'-TTCTTCCCACAGATAGAG-3' (SEQ ID NO: 1);

[0046] Primer-R: 5'-CTGGTCAGATCCCACAGAA-3' (SEQ ID NO: 2);

[0047] 2. Detection reagent: SYBR GreenMix.

[0048] Example 2

[0049] A detection method for quickly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles based on the detection kit described in Example 1

[0050] The samples to be tested are derived from peripheral blood samples of hematopoietic stem cell donors for HLA typing testing by the applicant. This embodiment has passed the review of the Medical Ethics Committee of Shenzhen Blood Center (SZBCMEC-2023-017), and the subjects have signed the informed consent for clinical research.

[0051] After extracting DNA from samples known to contain HLA-A*24:353 or HLA-A*24:02 by NGS sequencing, configure the amplification reaction system according to Table 1:

[0052] Table 1 qPCR amplification reaction system

[0053] name volume SYBRGreenMix 5μL Upstream primer (20 μM) 0.5μL Downstream primer (20 μM) 0.5μL Genomic DNA 2μL <![CDATA[ddH2O]]> 2μL Total volume 10μL

[0054] The sample loading layout of each reaction well is shown in Table 2.

[0055] Table 2 Reaction well layout

[0056]

[0057]

[0058] The amplification reaction program is shown in Table 3.

[0059] Table 3 Amplification reaction program

[0060]

[0061] After the PCR program is completed, the allele typing results are determined by observing the amplification curve and Ct value. If the sample to be tested has an amplification curve and a Ct value (less than 40), the sample contains the A*24:353 gene. If the sample to be tested fails to amplify and has no Ct value, the sample contains the A*24:02 gene.

[0062] The results of quantitative PCR of the samples are shown in Figure 1 and Table 4, and the corresponding amplification curves are shown in Figure 2 ; The red line indicates that the test sample has an amplification product, containing the HLA-A*24:353 allele; the green line indicates that the test sample has no amplification product, containing the HLA-A*24:02 allele.

[0063] Table 4 Detection results of different reaction wells

[0064] 1 2 3 4 5 6 7 8 9 10 A 35.17 36.85 33.95 35.37 35.28 35.29 37.45 35.91 36.20 37.39 B 35.54 35.94 36.69 35.65 C none none none none none none none none none none D none none none none none

[0065] Example 3

[0066] The SBT was used to test samples S1-S35 containing HLA-A*24:02 or HLA-A*24:353, and the HLA-A*24:353 positive control sample S36. After genomic DNA was extracted, the amplification reaction system was prepared using the reagents in the kit described in Example 1, as shown in Table 1. The layout of the reaction wells is shown in Table 5 below.

[0067] Table 5 Reaction well layout

[0068]

[0069]

[0070] The reaction program was: 95°C for 2 min; 95°C for 15 s, 55°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 2 min.

[0071] The results are shown in Table 6 and Figure 3 The corresponding amplification curve is shown in Figure 4 .

[0072] Table 6 Ct value results of each reaction well detection

[0073] 1 2 3 4 5 6 7 8 9 10 A none none none none none none none none none none B none none none none none none none none none none C none none none none none none none none none none D none none none none none 30.31

[0074] The results showed that the tested samples S1-S35 were all of HLA-A*24:02 genotype. The test results were later verified by NGS to be correct.

[0075] Comparative Example 1

[0076] The commercial kit produced by Thermo was used to distinguish the HLA-A*24:02 / HLA-A*24:353 alleles in the SBT test of samples containing HLA-A*24:02:01G. The detection method is shown in the operating instructions. Figure 5 .

[0077] The results show that the existing method for distinguishing A*24:02 and A*24:353 requires sequence primer specific amplification (amplification conditions are: 96℃130sec→63℃60sec→96℃10sec, 63℃60sec, 9Cycles→96℃10sec, 59℃50sec, 72℃30sec, 20Cycles→4℃ storage), followed by nucleic acid electrophoresis detection, and allele determination based on the presence or absence of the target band (indicated by the arrow). This prior art is time-consuming, the nucleic acid electrophoresis steps are cumbersome, and positive quality control is required. The results are interfered by other factors (including agarose concentration, fluorescent dye intensity, electrophoresis conditions, etc.), and the stability is poor.

[0078] Comparative Example 2

[0079] After DNA was extracted from samples known to contain HLA-A*24:353 or HLA-A*24:02 (same as in Example 2), an amplification reaction system was prepared according to the following composition: SYBR Green Mix 5 μL, upstream primer (20 μM) 0.5 μL, downstream primer (20 μM) 0.5 μL, genomic DNA 2 μL, ddH2O 2 μL, total volume 10 μL. The primer sequences are:

[0080] Upstream primer: 5'-TAGAGAAGGAGGGAGCTA-3' (SEQ ID NO: 3);

[0081] Downstream primer: 5'-CTGGTCAGATCCCACAGAA-3' (SEQ ID NO: 2).

[0082] The layout of the reaction wells is shown in Table 7 below:

[0083] Table 7 Reaction well layout

[0084] 1 2 3 4 5 6 7 8 9 10 A 353-1 353-2 353-3 353-4 353-5 353-6 353-7 353-8 353-9 353-10 B 353-11 353-12 353-13 353-14 C 02-1 02-2 02-3 02-4 02-5 02-6 02-7 02-8 02-9 02-10 D 02-11 02-12 02-13 02-14 02-15

[0085] The reaction procedure was: 95°C for 2 min; 95°C for 15 s, 61°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 2 min. The reaction results are shown in Table 8 and Figure 6 The amplification curve is shown in Figure 7 .

[0086] Table 8 Ct value detection results of different reaction wells

[0087] 1 2 3 4 5 6 7 8 9 10 A 23.57 23.30 23.54 23.45 25.90 24.21 23.56 23.09 25.85 23.54 B 24.06 23.94 25.56 24.28 C 35 35 35 35 35 35 35 35 35 33.37 D 33.50 35 35 35 35

[0088] The results showed that all samples had amplification products and the above primer pairs could not distinguish between the two alleles of HLA-A*24:353 and HLA-A*24:02.

[0089] Comparative Example 3

[0090] After DNA was extracted from samples known to contain A*24:353 or A*24:02 (same as in Example 2), an amplification reaction system was prepared according to the following composition: SYBR GreenMix 5 μL, upstream primer (20 μM) 0.5 μL, downstream primer (20 μM) 0.5 μL, genomic DNA 2 μL, ddH2O 2 μL, total volume 10 μL. The primer sequences were:

[0091] Upstream primer: 5'-TAGAGAAGGAGGGAGCTA-3' (SEQ ID NO: 3);

[0092] Downstream primer: 5'-CTGGTCAGATCCCACAGAA-3' (SEQ ID NO: 2).

[0093] The layout of the reaction wells is shown in Table 9 below:

[0094] Table 9 Reaction well layout

[0095] 1 2 3 4 5 6 7 8 9 10 A 353-1 353-2 353-3 353-4 353-5 353-6 353-7 353-8 353-9 353-10 B 353-11 353-12 353-13 353-14 C 02-1 02-2 02-3 02-4 02-5 02-6 02-7 02-8 02-9 02-10 D 02-11 02-12 02-13 02-14 02-15

[0096] The reaction program was: 95°C for 2 min; 95°C for 15 s, 62°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 2 min.

[0097] The reaction results are shown in Figure Figure 8 and Fig. 9 The corresponding Ct values ​​are shown in Table 10 below.

[0098] Table 10 Ct value detection results of different reaction wells

[0099] 1 2 3 4 5 6 7 8 9 10 A 25 25 25 25 25 25 25 25 25 25 B 25 25 none 25 C none none none none none none none none none none D none none none none none

[0100] The results showed that under this reaction condition, some samples would have false negative phenomenon, such as sample B3 was misjudged as A*24:02.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A detection primer for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, characterized in that: It includes a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

2.

2. A detection kit for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles based on fluorescent quantitative PCR technology, characterized in that: Comprising the detection primer according to claim 1.

3. The detection kit according to claim 2, characterized in that: Also includes SYBR Green Mix.

4. The detection kit according to claim 2 or 3, characterized in that: Also included are reagents for extracting sample genomic DNA.

5. Use of the detection primers according to claim 1 in preparing a rapid HLA-A*24:02 / HLA-A*24:353 allele detection kit.

6. A method for rapidly distinguishing HLA-A*24:02 / HLA-A*24:353 alleles, characterized in that: The following steps are involved: Using the genomic DNA of the sample to be tested as a template, the reaction system is configured using the detection primers described in claim 1, and qPCR detection is performed, and the typing result of the sample is determined according to the amplification curve of the sample to be tested and the presence or absence of a Ct value: When the sample to be tested has an amplification curve and the Ct value is ≤40, the sample to be tested contains the HLA-A*24:353 allele; When there is no amplification curve for the sample to be tested and the Ct value is greater than 40, the sample to be tested contains the HLA-A*24:02 allele.

7. The detection method according to claim 6, characterized in that: The reaction system is 10 μL, specifically including 5 μL of SYBR Green Mix, 0.5 μL of 20 μM upstream primer, 0.5 μL of 20 μM downstream primer, 2 μL of genomic DNA and 2 μL of ddH2O.

8. The detection method according to claim 7, characterized in that: The concentration of the genomic DNA is 25-200 ng / μL; the OD of the genomic DNA is 260 / OD 280 It is 1.65~1.

8.

9. The detection method according to claim 6, characterized in that: The reaction procedure of the qPCR detection is 95°C for 2 min; 95°C for 15 s, 55°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 2 min.

10. The detection method according to any one of claims 5 to 9, characterized in that: The sample to be tested includes at least one of the following: blood, tissue fluid and secretion fluid.

Images