FLUORESCENT PCR METHOD FOR DETECTING HLA-B*15:02 ALLELE AND SPECIFIC PRIMER PROBE COMBINATION THEREOF

Abstract

Disclosed is a fluorescent PCR method for detecting HLA-B*15:02 allele and a specific primer probe combination. In the present disclosure, a set of primers and probes are designed based on an HLA-B*15:02 specific SNP gene locus by using TaqMan probe technology, combining another set of primers and probes corresponding to the internal reference gene β-Actin, and a set of primer probe for non-HLA-B*15:02 genes are designed to detect whether a DNA sample contains an HLA-B*15:02 gene and whether a sample is homozygous or heterozygous. Compared with the similar detection methods in the past, the technical scheme in the present disclosure inherits the advantages of high specificity, high throughput, high resolution, low cost, simple and convenient operation, process controllability and the like of the fluorescent PCR, and may detect whether a sample is homozygous or heterozygous.

Claims

1. A specific primer probe combination for HLA-B*15:02 allele, comprising: HLA-B*15:02 allele-specific forward primer Fm, the nucleotide sequence of Fm is set forth in SEQ ID NO: 1 non-HLA-B*15:02 allele-specific forward primer Fn1, the nucleotide sequence of Fn1 is set forth in SEQ ID NO: 2 non-HLA-B*15:02 allele-specific forward primer Fn2, the nucleotide sequence of Fn2 is set forth in SEQ ID NO: 3 reverse primer R, the nucleotide sequence of R is set forth in SEQ ID NO: 4 probe P, the nucleotide sequence of P is set forth in SEQ ID NO. 5 wherein ROX is a fluorescent group, and BHQ1 is a quenching group.

2. The specific primer probe combination according to claim 1, further comprising specific primers and probes designed for an internal reference gene β-Actin, which are as follows: forward primer ACTB-F, the nucleotide sequence of ACTB-F is set forth in SEQ ID NO. 6 reverse primer ACTB-R, the nucleotide sequence of ACTB-R is set forth in SEQ ID NO: 7 probe ACTB-P, the nucleotide sequence of ACTB-P is set forth in SEO ID NO: 8 wherein FAM is a fluorescent group, and BHQ1 is a quenching group.

3. (canceled)

4. (canceled)

5. A kit for detecting HLA-B*15:02 allele by a fluorescent PCR reaction, comprising the specific primer probe combination according to claim 1.

6. A TaqMan probe real-time fluorescent PCR method for detecting HLA-B*15:02 allele for non-disease diagnostic purposes, comprising the following steps: 1) acquiring a genomic DNA of a sample to be tested; 2) adding the genomic DNA of the sample to be tested, a specific primer probe combination according to claim 1 and PCR related reaction reagents in a HLA-B*15:02 alleles positive reaction system and a HLA-B*15:02 alleles negative reaction system according to a ratio respectively; 3) detecting the reaction systems by fluorescence PCR, collecting fluorescence signals of the ROX and FAM fluorescence channels; 4) analyzing and judging the conditions of the HLA-B*15:02 allele carried by the sample to be tested by the fluorescence signal results.

7. The method according to claim 6, wherein the PCR related reaction reagents comprises dNTP, Taq DNA polymerase, PCR reaction buffer, and double distilled water for supplementing system.

8. The method according to claim 7, comprising the following components based on the reaction system of 20 μL: tube 1: 4 μL of 5×buffer, 2 μL of 10×buffer, 2 μL of dNTP, 0.4 μL of forward primer Fm, 0.4 μL of reverse primer R, 0.2 μL of probe P, 0.2 μL of internal reference forward primer, 0.2 μL of internal reference reverse primer, 0.1 μL of internal reference probe, 0.2 μL of Taq DNA polymerase, and 2 μL of genomic DNA of the sample to be tested, the volume is made up to 20 μL with double distilled water; tube 2: 4 μL of 5×buffer, 2 μL of 10×buffer, 2 μL of dNTP, 0.2 μL of forward primer Fn1, 0.2 μL of forward primer Fn2, 0.4 μL of reverse primer R, 0.2 μL of probe P, 0.2 μL of internal reference forward primer, 0.2 μL of internal reference reverse primer, 0.1 μL of internal reference probe, 0.2 μL of Taq DNA polymerase, and 2 μL of genomic DNA of the sample to be tested, the volume is made up to 20 μL with double distilled water; wherein, 5×buffer, 10×buffer and Taq DNA polymerase are from Hotstart HiTaq DNA Polymerase, a product of Fapon Biotech Inc.

9. The method according to claim 7, wherein a PCR amplification program is: Hold Stage: 50° C. 2 min; 95° C. 10 min; PCT Stage: 35 cycles in total: 95° C. for 15 s; 60° C. for 35 s.

10. The method according to claim 7, wherein a basis for judging the final detection result is as follows: specific primers and probes of a target gene and an internal reference gene are amplified in a double tube on a fluorescent PCR instrument, and the fluorescence is collected through corresponding channels; the internal reference gene is used as the quality control, and a fluorescence amplification curve must be displayed, otherwise the result is regarded as invalid; on the premise of successful amplification of the internal reference gene: if there is no amplification curve for HLA-B*15:02 allele positivity while there is an amplification curve for HLA-B*15:02 allele negativity, the sample is homozygous for HLA-B*15:02 allele negativity; if there is a fluorescent signal corresponding to the forward primer for the HLA-B*15:02 positive allele, the sample is HLA-B*15:02 positive; on the basis of HLA-B*15:02 positivity, the sample is an HLA-B*15:02 positive heterozygote if there is an amplification curve for HLA-B*15:02 allele negativity, otherwise, the sample is an HLA-B*15:02 positive homozygote.

11. The kit according to claim 5, wherein the specific primer probe combination according to claim 1, further comprising specific primers and probes designed for a internal reference gene β-Actin, which are as follows: forward primer ACTB-F, the nucleotide sequence of ACTB-F is set forth in SEQ ID NO: 6; reverse primer ACTB-R, the nucleotide sequence of ACTB-R is set forth in SEQ ID NO: 7; probe ACTB-P, the nucleotide sequence of ACTB-P is set forth in SEQ ID NO: 8; wherein FAM is a fluorescent group, and BHQ1 is a quenching group.

12. The TaqMan probe real-time fluorescent PCR method according to claim 6, wherein the specific primer probe combination according to claim 1, further comprising specific primers and probes designed for an internal reference gene β-Actin, which are specifically as follows: forward primer ACTB-F, the nucleotide sequence of ACTB-F is set forth in SEQ ID NO: 6; reverse primer ACTB-R, the nucleotide sequence of ACTB-R is set forth in SEQ ID NO: 7; probe ACTB-P, the nucleotide sequence of ACTB-P is set forth in SEQ ID NO: 8; wherein FAM is a fluorescent group, and BHQ1 is a quenching group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 shows the partial alignment results by sequence alignment of HLA-B*15:02 as a reference gene with other HLA-B alleles.

[0053] FIG. 2 shows the results of a control experiment using two polymerases.

[0054] FIG. 3 is a schematic diagram showing the results of introducing a different mismatched base to one base forward of the rs144012689 locus.

[0055] FIG. 4 is a schematic diagram of dilution results of a positive standard product in an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0056] The development process of the present disclosure is briefly described below, and the scheme of the present disclosure is further described in detail by examples.

[0057] It should be recognized that the following description of the development process of the present disclosure only selects part of the salient points, intending to show that the final scheme of the present disclosure is not available to those skilled in the art from a simple and limited experiments.

[0058] The HLA-B allele sequences are obtained from the Immuno Polymorphism Database (IPD) of EMBL-EBI, HLA-B*15:02 is used as the reference gene for sequence alignment with other HLA-B alleles, and the alignment results are obtained, some results are shown in FIG. 1. In which, each row represents each HLA-B allele, and each column represents the comparison of each allele with the HLA-B:15*02 gene at each locus, “-” represents a match, and “.” represents a vacancy, and the bases “A”, “T”, “C” and “G” represent the corresponding bases which are different from the HLA-B:15*02 gene at the locus and are marked. For example, the base of HLA-B*07:02:01:03 at −262 is T, while the base of HLA-B:15*02 is C at this locus, which can be used as the basis for finding specific loci.

[0059] According to experience, simultaneous differentiation of the HLA-B:15*02 gene with as many other genes as possible is achieved by multiple specific loci simultaneous detection. Therefore, the data are processed, the result of sequence alignment is first converted into a matrix, where the vacancy “.” and the same base “-” are represented by 0, indicating that the locus could not identify HLA-B:15*02 and the corresponding allele; the results of mismatching, that is, “A”, “T”, “C” and “G”, are represented by 1, indicating that this locus may identify HLA-B:15*02 and the corresponding allele, i.e., this locus is the specific locus for identifying HLA-B:15*02 and the corresponding allele.

TABLE-US-00003 TABLE 1 — — A G — — G — — ↓ 0 0 1 1 0 0 1 0 0

[0060] As shown in Table 1, the locus represented in the first column may distinguish the HLA-B:15*02 gene from the alleles represented in the second row and the third row, and the locus represented in the third column may distinguish the HLA-B:15*02 gene from the alleles represented in the first row, then detecting the loci represented in the first column and the third column at the same time may distinguish the HLA-B:15*02 gene from the three alleles simultaneously. According to this idea, the comparison results are converted into a matrix containing only “1” and “0”, and the number of non-zero elements in each column vector is the number of alleles that can be distinguished at that locus. As shown in the above table, the number of non-zero elements in the first column is 2, and the number of non-zero elements in the third column is 1, which indicates that the number of alleles that may be distinguished at the corresponding loci in the first column and the third column are 2 and 1. On this basis, the number of non-zero elements in the obtained result by adding multiple column vectors is the number of alleles that may be distinguished by simultaneous detection of multiple loci, as shown in the above table, [0,1,1] plus [1,0,0] equals to [1,1,1], wherein the number of non-zero elements is 3, which means that simultaneous detection of the corresponding loci in the first column and the third column may simultaneously distinguish the HLA-B:15*02 gene from three alleles.

[0061] According to the above method, the whole sequence is screened for specific locus combination, and the optimal result is the combination of rs1050388, rs151341118, rs1050692, rs41540133 and rs144012689. The number of alleles that could be distinguished among the 3191 HLA-B alleles is 3191, and the degree of distinction is 100%. Each locus in the combination is analyzed, the number of alleles that could be distinguished from rs144012689 is 3187, and the number of alleles that could be distinguished from the remaining four specific loci is 1, in which, the distinguishable allele for rs1050388 is HLA-B*15:13:01, the distinguishable allele for rs151341118 is HLA-B*15:438, the distinguishable allele for rs1050692 is HLA-B*15:454N, the distinguishable allele for rs41540133 is HLA-B*15:491. The four alleles are investigated in the Allele Frequency Net Database (AFND) and found that the gene frequencies in China are all below 0.01%, and thus specific primer probes only for rs144012689 are designed.

[0062] Analyzing the forward and reverse sequences of rs144012689, another specific locus rs2596496 is found at two bases forward. Therefore, rs144012689 may be used as the 3′ end of the forward primer, and the combination of multiple primers and single probe combined with the Amplification Refractory Mutation System (ARMS) method may not only identify the HLA-B:15*02 gene, but also determine the heterozygosity of the HLA-B:15*02 gene in the sample through the heterozygosity of rs144012689.

[0063] The reagents frequently used in the past are selected for experiments, and it is found that the previously used TakaraPremix Ex Taq kit has the disadvantages of high amplification efficiency and poor specificity, and severe non-specific amplification would occur. Therefore, in combination with the product investigation on related polymerases,

[0064] HotstartHiTaq DNA Polymerase is finally used. Two enzymes are used to conduct control experiments on negative and positive samples, and the results are shown in FIG. 2. The first two columns are the results of using Takara Premix Ex Taq, the last two columns are the results of using HotstartHiTaq DNA Polymerase, columns 1 and 3 are for positive samples, and columns 2 and 4 are for negative samples. In the results of this experiment, a false-positive result is obtained for Takara Premix Ex Taq, while the results for HotstartHiTaq DNA Polymerase are accurate.

[0065] Based on the Amplification Referential Mutation System (ARMS) method, a mismatched base is artificially introduced into one base forward of the rs144012689 locus, while the correctly matched base is C, therefore the primers with three bases (A, G and T) at the corresponding loci are designed and experimented, and the results are shown in FIG. 3. In which, according to the CT value from small to large, the corresponding bases at the ARMS locus are C, A, G and T. Because the C\G and C\T mismatches are too strong, resulting in an excessive decrease in amplification efficiency, which could result in a false negative result, the final ARMS mismatch base is determined to be A.

[0066] An example of the present disclosure will be described below.

[0067] 1. Preparation of Samples

[0068] DNA was extracted from peripheral blood samples using a QIAamp DNA Mini Blood Kit (Qiagen, Germany).

[0069] 2. Design of Primers

[0070] The specific primers and probes for the HLA-B*15:02 gene were as follows:

TABLE-US-00004   forward primer Fm: (SEQ ID NO: 1) 5′-CCTCATTACTGGGAAGCAGCATCAT-3′; reverse primer R: (SEQ ID NO: 4) 5′-CCACAACCATCAAGGCGATACATCT-3′; Probe P: (SEQ ID NO: 5) 5′-ROX-ACGCAGCCTGGGACCCTGTGTGCCAGCA-BHQ1-3′.

[0071] The specific primers and probes for the non-HLA-B*15:02 genes were as follows:

TABLE-US-00005   forward primer Fn1: (SEQ ID NO: 2) 5′-CCTCATTACTGGGAAGCAGCATCAA-3′; forward primer Fn2: (SEQ ID NO: 3) 5′-CCTCATTACTGGGAAGCAGCATGAA-3′; reverse primer R: (SEQ ID NO: 4) 5′-CCACAACCATCAAGGCGATACATCT-3′; Probe P: (SEQ ID NO: 5) 5′-ROX-ACGCAGCCTGGGACCCTGTGTGCCAGCA-BHQ1-3′;

[0072] wherein ROX was Carboxy-X-RhoDamine; BHQ1 was Black Hole Quencher-1.

[0073] The primers and probes for the internal reference gene ACTB were as follows:

TABLE-US-00006   forward primer ACTB-F: (SEQ ID NO: 6) 5'-CTCTCTGACTAGGTGTCTAAGACA-3'; reverse primer ACTB-R: (SEQ ID NO: 7) 5'-GAGTCTGTTCAGACCTACTGTG-3'; Probe ACTB-P: (SEQ ID NO: 8) 5'-FAM-AGGTACTAACACTGGCTCGTGTGAC-BHQ1-3';

[0074] wherein FAM was 6-carboxy-fluorescein, and BHQ1 was Black Hole Quencher-1.

[0075] 3. Sample Detection

[0076] The reaction system was 20 μL, including:

[0077] Tube 1: 44, of 5×buffer, 2 μL of 10×buffer, 2 μL of dNTP, 0.4 μL of forward primer Fm, 0.4 μL of reverse primer R, 0.2 μL of probe P, 0.2 μL of internal reference forward primer, 0.2 μL of internal reference reverse primer, 0.1 μL of internal reference probe, 0.2 μL of Taq DNA polymerase, and 2 μL of genomic DNA of the sample to be tested, the volume is made up to 20 μL with double distilled water.

[0078] Tube 2: 44, of 5×buffer, 2 μL of 10×buffer, 2 μL of dNTP, 0.2 μL of forward primer Fn1, 0.2 μL of forward primer Fn2, 0.4 μL of reverse primer R, 0.2 μL of probe P, 0.2 μL of internal reference forward primer, 0.2 μL of internal reference reverse primer, 0.14, of internal reference probe, 0.2 μL of Taq DNA polymerase, and 2 μL of genomic DNA of the sample to be tested, the volume is made up to 20 μL with double distilled water.

[0079] The PCR amplification program was:

[0080] Hold Stage: 50° C. 2 min; 95° C. 10 min;

[0081] PCT Stage (35 cycles) 95° C. for 15 s; 60° C. for 35 s.

[0082] 4. Analysis of Results

[0083] The internal reference gene was used as the quality control, and a fluorescence amplification curve must be displayed, otherwise the result was regarded as invalid; on the premise of successful amplification of the internal reference gene, if there was no amplification curve for HLA-B*15:02 allele positivity while there was an amplification curve for HLA-B*15:02 allele negativity, the sample was homozygous for HLA-B*15:02 allele negativity. If there was a fluorescent signal corresponding to the forward primer for the HLA-B*15:02 positive allele, the sample was HLA-B*15:02 positive; on the basis of HLA-B*15:02 positivity, the sample was an HLA-B*15:02 positive heterozygote if there was an amplification curve for HLA-B*15:02 allele negativity, otherwise, the sample was an HLA-B*15:02 positive homozygote.

[0084] 5. Experimental Results

[0085] The ROX channel signal of the positive standard dilution result was shown in FIG. 4, from which a recommended sample concentration was 10 ng/μL.

[0086] Verification experiment: 100 random samples were amplified, and the results were compared with those of SBT sequencing (Beijing BoFurui Medical Laboratory Co., Ltd.) to determine the accuracy and sensitivity of the method. The results were shown in Table 2, and the accuracy and sensitivity of this method were both 100%.

TABLE-US-00007 TABLE 2 Results compared with SBT sequencing method SBT sequencing Positive Positive Neg- homozygosity heterozygosity ative Total PCR Positive 1  0  0  1 results homozygosity Positive 0 10  0  10 heterozygosity Negative 0  0 89  89 Total 1 10 89 100

[0087] The method for detecting the HLA-B*15:02 genotype by single gene locus provided in the example has the advantages of high efficiency and accuracy, good specificity and simple operation process, which is beneficial to guiding the safe medication of carbamazepine on the basis of HLA-B*15:02 allele typing.

[0088] In addition, base on the above specific primer probe combinations, by using the existing technical means, detection tools, such as a kit and a chip, for detecting the HLA-B*15:02 allele based on the fluorescent PCR reaction may be prepared accordingly.