Kit for detecting HLA (human leukocyte antigen) genotype by fluorescent PCR (polymerase chain reaction) melting curve method

Abstract

The invention provides a kit for detecting HLA (human leukocyte antigen) genotypes by a fluorescent PCR (polymerase chain reaction) melting curve method, which comprises genotypes of HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1, amplification is performed by a 96-well optical reaction plate containing primers, double-strand DNA (deoxyribonucleic acid) products obtained by amplification are actively combined by SYBR Green I, low-resolution genotypes of HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 are comprehensively judged according to results of a 96-well melting curve, and high-resolution genotypes of HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 are obtained. Therefore, matching diagnosis of organ transplantation is assisted.

Description

[0001] This application is a divisional application with the application date of July 19, 2017, application number 201710589013.0, and the invention name is "A kit for detecting HLA genotype by fluorescent PCR melting curve method". Technical Field

[0002] The invention belongs to the field of fluorescent qualitative PCR, and in particular relates to a kit for detecting HLA genotypes by a fluorescent PCR melting curve method. Background Art

[0003] Antigenic substances distributed on the surface of nucleated cells of organisms encoded by the major histocompatibility complex are called human leukocyte antigens (HLA) for humans. The HLA molecule genes located on the short arm of the sixth pair of human chromosomes have a high degree of diversity. After the decryption of the HLA molecule gene sequence, it was found that most HLAs differed by only a few or even only one amino acid. Therefore, the previous serological methods were usually unable to effectively identify and distinguish HLA types like nucleic acid detection methods.

[0004] According to the research findings over the years, almost all blood cells and tissue cells have the expression of HLA antigens. This antigen, which represents individual specificity, is the main substance that causes immune rejection during organ transplantation. The HLA antigen type is formed by complex gene arrangements and their molecular products, involving immune regulation and cell differentiation. It is the most complex genetic polymorphism system in the human body. HLA antigens include A, B, C, D (DR, DQ, DP) E, F and other genes, among which A, B, DR, DQ are the typing genes locked by this product.

[0005] In the past, HLA-ABDRDQ was mainly tested using serological methods, which involved many steps and was subjective in the interpretation of results. The quality of serum, complement, and isolated lymphocytes would affect the accuracy of the results. After the gene sequence was decrypted, it was found that there were only very few differences between most types, which could not be presented one by one by serology. As a result, nucleic acid testing based on polymerase chain reaction (PCR) has gradually become one of the mainstream methods for detecting HLA antigens. Summary of the invention

[0006] The object of the present invention is to provide a kit for detecting HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 genotyping by a fluorescent PCR melting curve method.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The present invention provides a primer set for detecting HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 genotyping by a fluorescent PCR melting curve method, comprising: specific primers having sequences as shown in SEQ ID NO.1-256.

[0009] The primer set of the present invention also includes internal control primers whose sequences are shown as SEQ ID NOs. 257-262.

[0010] The primer sequences in the primer set of the present invention are:

[0011] Table 1 Primer sequences:

[0012]

[0013]

[0014]

[0015] The primer pairs were distributed in a 96-well plate as follows:

[0016]

[0017]

[0018] Furthermore, the present invention provides a kit for detecting HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 genotyping using a fluorescent PCR melting curve method, which comprises the primer set described above.

[0019] The present invention includes HLA-A, HLA-B, HLA-DRB1, HLA-DQB genotyping detection and internal quality control monitoring in a 96-well optical reaction plate of a kit. The double-stranded DNA product generated after PCR amplification can be combined with SYBRGreen I. The double-stranded DNA melting temperatures (Tm values) of different products are different. After low-temperature to high-temperature melting curve analysis, when the double strands dissociate to their Tm values, the fluorescence will drop significantly, thereby distinguishing specific products. By detecting the results of the 96-well melting curve analysis, the low-resolution genotyping of HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 is comprehensively judged, thereby assisting the matching diagnosis of organ transplantation.

[0020] The kit contains 4 components: optical reaction plate, T3 reaction solution, nucleic acid polymerase, optical sealing film,

[0021] In the present invention, the optical reaction plate comprises 262 specific primers, wherein No. 1 to No. 256 are specific primers, and the sequences are shown in SEQ ID NO. 1 to No. 256, and No. 257 to No. 262 are internal control primers, and the sequences are shown in SEQ ID NO. 257 to No. 262;

[0022] In the present invention, the nucleic acid polymerase comprises Taq DNA polymerase; preferably, the nucleic acid polymerase formula is: Taq enzyme 5U / μL.

[0023] In the present invention, the T3 reaction solution includes a buffer solution containing a relevant PCR reaction; preferably, the T3 reaction solution #formula is: 50mM KCl, 20mMTris-HCl, 2.0mM MgCl 2 , 0.2mM dNTP, SYBR green I 0.1×, ROXreference dye 30nM.

[0024] The optical sealing film is a medical grade polymer with a size of 13*8cm 2 .

[0025] The present invention also provides a method for detecting HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 genotyping, which comprises detecting a sample with the primer set as described above or with the kit as described above. The method described in the present invention may be for diagnostic purposes or non-diagnostic purposes, and the present invention is not limited thereto.

[0026] Preferably, the detection adopts fluorescent PCR method, and the detection channels are selected as FAM and ROX.

[0027] Amplification conditions included:

[0028] 96℃ for 2 minutes and 30 seconds; enter the following cycle: 96℃ for 15 seconds, 65℃ for 1 minute, for a total of 10 cycles; enter the following cycle: 95℃ for 15 seconds, 62℃ for 50 seconds, 72℃ for 30 seconds, for a total of 22 cycles; enter melting curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, perform signal collection at a heating rate of 1% from 60℃ to 95℃, 95℃ for 15 seconds, 60℃ for 15 seconds.

[0029] The advantages of the present invention are:

[0030] Advantages: The advantages of the present invention are: 1. After the fluorescent PCR program is completed, it can be directly interpreted without the need for subsequent experimental operations, and the detection only takes 2 hours; 2. It includes the genotypes required for conventional organ transplantation, such as HLA-A, HLA-B, HLA-DRB1, HLA-DQB, etc.

[0031] Beneficial effects: 1. Provide rapid result reference for clinical organ transplantation; 2. One test covers the HLA loci for routine clinical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a typical negative result of melting curve analysis; the Tm value is 77.32℃, which is in line with the negative judgment range (75~79.5℃);

[0033] Figure 2 This is a typical positive result of melting curve analysis; the Tm value is 89.56°C, which is in line with the positive judgment range (87-92°C);

[0034] Figure 3 This is a typical positive result of melting curve analysis; the Tm value is 86.62°C, which is in line with the positive judgment range (84-89°C);

[0035] Figure 4 Typical positive results of melting curve analysis; Tm values ​​are 86.5℃ and 90.15℃, which are in line with the positive judgment range (83.5-87.9℃ and 88-92℃);

[0036] Figure 5 The positive and negative results obtained from the test of sample 1;

[0037] Figure 6 HLA-A gene interpretation table;

[0038] Figure 7 HLA-B gene interpretation table;

[0039] Figure 8 HLA-DRB1 gene interpretation table;

[0040] Fig. 9 HLA-DQB1 gene interpretation table. DETAILED DESCRIPTION

[0041] Example 1

[0042] 1. Preparation of reaction system

[0043] (1) Preparation of optical reaction plate

[0044] Take a 10mL volumetric flask, add 1500μL of 0.5OD / mL specific primer and 375μL of 0.5OD / mL internal quality control primer specified in the 96-well formula table, make up the volume to 10mL with sterile water, turn it over to mix it thoroughly, transfer the liquid to a 15mL centrifuge tube, use an automated pipetting workstation to dispense it into a 96-well deep-well raw material plate, and then use an original plate-type dispensing workstation to dispense the primer mixture in the raw material plate into the optical reaction plate. After a brief centrifugation, dry it in an environment with a temperature of 22±2℃ and a humidity of 10±5%RH for 16-24 hours. After sealing in a bag, it is a usable optical reaction plate and stored at -20℃ for later use.

[0045] Table 2 Primer arrangement on 96-well plate

[0046]

[0047]

[0048] The genotypes of each well corresponding to the above primer settings are shown in Table 3:

[0049] Table 3 Genotypes corresponding to 96-well plates

[0050]

[0051]

[0052] (2) Preparation of T3 reaction solution

[0053] Take a 100mL volumetric flask and add 0.833mL of 6M KCl, 2mL of 1M Tris-HCl, and 1MMgCl 2 0.2mL, 100mM dNTP 0.2mL, 10×SYBR Green I 1mL, 25μM ROX reference dye 0.12mL. Make up the volume to 100mL with double distilled water, mix thoroughly with a rotor, dispense into centrifuge tubes at 1.53mL / tube, and store at -20℃ for later use.

[0054] 2. Nucleic acid extraction

[0055] Use a registered nucleic acid extraction kit to extract nucleic acid. After extraction, use a micro-UV spectrophotometer to measure the purity of the nucleic acid. The OD260 / OD280 should be between 1.6 and 2.0.

[0056] 3. Load the sample onto the machine

[0057] (1) Preparation of reaction mixture

[0058] Take out the optical reaction plate and thaw the T3 reaction solution, take out the nucleic acid polymerase and keep it on the ice box, take 9.2 μL of nucleic acid polymerase and add it to the T3 reaction solution, shake and mix, and centrifuge briefly.

[0059] (2) Sample addition

[0060] Take 15 μL of the above mixed solution and dispense it into the A1 reaction well of the ABDRDQ reaction plate, add 3 μL of sterilized ultrapure water to the A1 well, then take 303 μL of the extracted sample and add it to the mixture of T3 reaction solution, shake and mix, centrifuge briefly, and dispense 18 μL into the remaining reaction wells of the optical reaction plate. After sealing with optical sealing film, centrifuge briefly, and immediately perform PCR amplification reaction.

[0061] (3) On-machine testing

[0062] 1) Loop condition setting

[0063] 96℃ for 2 minutes and 30 seconds; enter the following cycle: 96℃ for 15 seconds, 65℃ for 1 minute, for a total of 10 cycles; enter the following cycle 95℃ for 15 seconds, 62℃ for 50 seconds, 72℃ for 30 seconds (signal collection), for a total of 22 cycles; enter melting curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, perform signal collection from 60℃ to 95℃ at a heating rate of 1%, 95℃ for 15 seconds, 60℃ for 15 seconds.

[0064] 2) Instrument detection channel selection

[0065] Fluorescent markers were set to FAM and ROX.

[0066] 4. Result judgment

[0067] (1) Well A1 does not have a Tm value of 75-79.5; (2) The negative and positive judgments for each well should refer to the judgment value range in the table below; (3) The Applied Biosystems 7500 instrument chooses to transfer to a TXT file containing Results and Multicomponent Data, and then imports it into the HLA-ABDRDQ result analysis software for analysis to directly obtain the HLA allele typing test results.

[0068] Table 4 Negative and positive judgment criteria for each well

[0069]

[0070]

[0071] Example 2

[0072] 1. Reagent specificity verification

[0073] (1) Experimental samples

[0074] Six specific samples were taken to verify the specificity of the reagent, and the samples were purchased from the internationally recognized IHWG (http: / / www.ihwg.org / cellbank / ) standards.

[0075] (2) Experimental process

[0076] The above 6 specific samples were tested separately using the HLA-ABDRDQ (fluorescence PCR melting curve method) nucleic acid detection kit, and the test results were analyzed to verify the specificity of the reagent.

[0077] (3) Experimental results

[0078] The test results of the 6 specific samples were all in line with expectations, indicating that the reagents had good specificity and no cross-reaction. The specific results are shown in the table below:

[0079] Table 5 Specificity detection results

[0080]

[0081] 2. Verification of reagent precision

[0082] (1) Experimental samples

[0083] One precision sample (concentration of 10 ng / μL) was taken to verify the precision of the reagent. The sample was purchased from the internationally recognized IHWG (http: / / www.ihwg.org / cellbank / ) standard.

[0084] (2) Experimental process

[0085] The above precision samples were tested 10 times using the HLA-ABDRDQ (fluorescence PCR melting curve method) nucleic acid detection kit, and the test results were analyzed to verify the precision of the reagent.

[0086] (3) Experimental results

[0087] The coefficient of variation (CV value) of the precision samples within the batch is less than 5%, indicating that the reagent has good repeatability. The specific results are shown in the table below:

[0088] Table 6 Precision test results

[0089]

[0090]

[0091]

[0092]

[0093] 3. Verification of the minimum detection limit of reagents

[0094] (1) Experimental samples

[0095] Five clinical samples of different genotypes were taken and the nucleic acid concentration was diluted to 10 ng / μL for amplification to verify the minimum detection limit of the reagent.

[0096] (2) Experimental process

[0097] The above minimum detection limit was repeatedly tested 10 times using the HLA-ABDRDQ (fluorescence PCR melting curve method) nucleic acid detection kit, and the test results were analyzed to verify the minimum detection limit of the reagent.

[0098] (3) Experimental results

[0099] The minimum detection limit verification results are consistent with the expected type, and the minimum detection limit of the reagent is 10ng. The specific results are shown in the table below:

[0100] Table 7 Minimum detection limit

[0101] Sample No. 1 2 3 4 5 Minimum detection limit 10ng 10ng 10ng 10ng 10ng

[0102] Example 3: Results of testing 30 clinical samples

[0103] 1. According to the preparation method shown in Example 1, prepare the relevant components of the kit and store them at -20°C for future use.

[0104] 2. The genomic DNA of 30 clinical samples was extracted using the nucleic acid extraction reagent (whole blood type) produced by TBG Biotech (Xiamen) Co., Ltd. The purity of the DNA samples was detected using an ultraviolet spectrophotometer. The OD260 / OD280 of the 30 samples were all between 1.6 and 2.0.

[0105] 3. According to the steps shown in Example 1, DNA was added and tested on a fluorescent quantitative PCR instrument. The instrument used this time was ABI7500.

[0106] 4. According to the judgment criteria shown in Example 1, the results are interpreted and counted.

[0107] Take the interpretation of sample 1 as an example:

[0108] HLA-A gene:

[0109] like Figure 5 As shown, sample 1 was tested using the kit and the results of Example 1 obtained the following 96-well positive and negative results, with the yellow background indicating that there were two positive Tm values ​​in the wells, and the green background indicating that the positive Tm value results appeared in the wells.

[0110] Next, according to the HLA-A gene interpretation table ( Figure 6) The positive and negative results showed that HLA-A*24 and HLA-A*31 were the combined genotype test results of sample 1. Some wells had two positive Tm values, and the green ones were the wells with positive results for the corresponding genotypes.

[0111] According to the HLA-B gene interpretation table ( Figure 7 ) The positive and negative results are obtained, HLA-B*07 and HLA-B*40 are the combination of genotype test results of sample 1 (the yellow background is the well position with two positive Tm values, and the green is the well position with positive Tm value results of the corresponding genotype)

[0112] According to the HLA-DRB1 gene interpretation table ( Figure 8 ) The positive and negative results show that HLA-DRB1*03 and HLA-DRB1*07 are the combined results of the genotype detection of sample 1 (the yellow background indicates that there are two positive Tm values ​​in the wells, and the green background indicates that the corresponding genotype has a positive Tm value result well)

[0113] Then according to the HLA-DQB1 gene interpretation table ( Fig. 9 ) The positive and negative results obtained were HLA-DQB1*03 and HLA-DQB1*03 as the combination of genotype detection results for sample 1 (the yellow background indicates that there are two positive Tm values ​​in the wells, and the green background indicates the wells with positive Tm value results for the corresponding genotypes).

[0114] According to the process demonstrated above, the genotype of sample 1 can be obtained as follows: HLA-A24, HLA-A31, HLA-B40, HLA-B 07, HLA-DRB1 03, HLA-DRB107, HLA-DQB 03, HLA-DRB1 03.

[0115] The genotype identification results of 30 clinical samples are shown in Table 8.

[0116] Table 8 Genotyping results of 30 clinical samples

[0117]

[0118]

[0119] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A primer set for detecting HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 genotyping by fluorescent PCR melting curve method, comprising: The specific primers have sequences as shown in SEQ ID NO.1-256.

2. The primer set according to claim 1, characterized in that Also included are internal control primers whose sequences are shown as SEQ ID NOs. 257-262.

3. The primer set according to claim 1 or 2, characterized in that: The primer pairs were distributed in a 96-well plate as follows:

4. A kit for detecting HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 genotyping by fluorescent PCR melting curve method, comprising the primer set according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that include: Optical reaction plate, optical sealing film, T3 reaction solution and nucleic acid polymerase; The optical reaction plate comprises 262 specific primers, wherein primers 1 to 256 are specific primers, and the sequences are shown in SEQ ID NO.1-256, and primers 257 to 262 are internal control primers, and the sequences are shown in SEQ ID NO.257-262; The T3 reaction solution includes a buffer containing a relevant PCR reaction; The nucleic acid polymerase comprises Taq DNA polymerase.

6. The kit according to claim 5, characterized in that The T3 reaction solution includes 50mM KCl, 20mM Tris-HCl, 2.0mM MgCl2, 0.2mM dNTP, SYBR greenI 0.1×, and ROX reference dye 30nM.

7. The kit according to claim 5, characterized in that The nucleic acid polymerase includes 5U / μL Taq DNA polymerase.

8. The kit according to claim 5, characterized in that The optical sealing film is a medical grade polymer with a size of 13*8cm 2 .

9. A method for detecting HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 genotyping for non-diagnostic purposes, comprising detecting a sample using the primer set described in any one of claims 1 to 3 or the kit described in any one of claims 4 to 8.

10. The method according to claim 9, characterized in that The detection adopts fluorescent PCR method, and the detection channels are selected as FAM and ROX; the amplification conditions include: 96℃ for 2 minutes and 30 seconds; enter the following cycle: 96℃ for 15 seconds, 65℃ for 1 minute, for a total of 10 cycles; enter the following cycle: 95℃ for 15 seconds, 62℃ for 50 seconds, 72℃ for 30 seconds, for a total of 22 cycles; enter melting curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, perform signal collection at a heating rate of 1% from 60℃ to 95℃, 95℃ for 15 seconds, 60℃ for 15 seconds.

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