CN117187374A - Qualitative detection kit for HLA-B5801 allele - Google Patents
Qualitative detection kit for HLA-B5801 allele Download PDFInfo
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Abstract
The invention discloses a qualitative detection kit for HLA-B5801 alleles. The invention provides a primer and probe combination capable of detecting HLA-B5801 allele based on a double-start oligonucleotide primer and a fluorescent PCR technology aiming at HLA-B5801 allele. On the basis, the invention provides a qualitative detection kit and a detection method for HLA-B5801 alleles by matching with reagents required by PCR reactions such as hot-start DNA polymerase and the like. The primer and probe combination can specifically amplify HLA-B5801 alleles, can accurately detect the HLA-B5801 alleles, has the advantages of short detection time, high specificity and sensitivity, low cost and the like, and is beneficial to personalized medicine application of allopurinol.
Description
Technical Field
The invention belongs to the technical field of gene detection. In particular to a qualitative detection kit for HLA-B5801 alleles.
Background
Allopurinol (Allopurinol) is a drug capable of effectively controlling uric acid, and is widely applied to the treatment of gout, hyperuricemia, chronic kidney disease accompanied with hyperuricemia and the like. It was found that the population carrying the HLA-B5801 allele (HLA-B5801 gene positive) was prone to onset of the stervens-Johnson syndrome (SJS) and toxic epidermolysis necrotica (Toxic epidermal necrolysis, TEN) when allopurinol was used. Since Shi Difen syndrome and toxic epidermonecrosis are serious allergic diseases, systemic damage can be caused, and death of patients can be caused when serious. Therefore, screening of HLA-B5801 allele is necessary before allopurinol is used.
Human leukocyte antigens (Human leukocyte antigens, HLA) are expression products of human histocompatibility complexes, are widely expressed on the surface of nucleated cells of various tissues, and comprise HLA-A, -B, -C and other series of antigens, and a plurality of alleles with high homology with HLA-B5801 exist at the B gene locus. In actual detection, each homologous allele was susceptible to cross-reactivity with HLA-B5801. Therefore, reagents for detecting HLA-B5801 need to have extremely strong specificity. Although there are many reports of HLA-B5801 allele detection products and methods, it is not clear whether HLA-B5801 can be distinguished from other alleles with high homology, and specific detection can be achieved. The product for detecting HLA-B5801 alleles with high specificity and sensitivity is of great significance for guiding individual administration of allopurinol.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide a qualitative detection kit for HLA-B5801 alleles.
The first object of the present invention is to provide a primer and probe combination for qualitative detection of HLA-B5801 allele.
A second object of the present invention is to provide the use of said primer and probe combination for the preparation of a qualitative detection product for HLA-B5801 allele.
The third object of the invention is to provide a qualitative detection kit for HLA-B5801 allele.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a primer and probe combination for detecting HLA-B5801 allele based on dual priming oligonucleotide primer (dual priming oligonucleotide, DPO) and fluorescent PCR technology. On the basis, the invention provides a qualitative detection kit and a detection method for HLA-B5801 alleles by matching with reagents required by PCR amplification such as hot-start DNA polymerase and the like. The primer and probe combination can be used for specifically amplifying HLA-B5801, but not amplifying other highly homologous alleles, can accurately detect the HLA-B5801 allele, and has the advantages of short detection time, high specificity, simple and visual interpretation, low cost and the like.
The invention provides a primer and probe combination for qualitatively detecting HLA-B5801 allele based on a double-start oligonucleotide primer and a fluorescent PCR technology, which comprises a primer 5801-F/R and a probe 5801-P; the nucleotide sequence of the primer 5801-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer 5801-R is shown as SEQ ID NO.2, and the nucleotide sequence of the probe 5801-P is shown as SEQ ID NO. 5.
The primer 5801-F contains IIIII besides the base A/T/C/G, wherein IIIII is 5 hypoxanthine or deoxyhypoxanthine.
Specifically, the primer and probe combination of the invention also comprises an internal standard primer GAPDH-DF2/DR2 and an internal standard probe GAPDH-DP2; the nucleotide sequence of the internal reference primer GAPDH-DF2 is shown as SEQ ID NO.3, the nucleotide sequence of the internal reference primer GAPDH-DR2 is shown as SEQ ID NO.4, and the nucleotide sequence of the internal reference probe GAPDH-DP2 is shown as SEQ ID NO. 6.
Specifically, the probe of the invention is also marked with different fluorescent groups.
As an alternative embodiment, the 5 'end of the probe 5801-P of the invention is marked with FAM, and the 3' end is marked with BHQ1; the probe GAPDH-DP2 is labeled with CY5 at the 5 'end and BHQ2 at the 3' end.
The primer and probe combination can be used for sensitively and specifically detecting HLA-B5801 alleles. Thus, the use of said primer and probe combinations for the preparation of a qualitative detection product of HLA-B5801 allele is claimed.
Optionally, the product is a kit.
The invention also provides a qualitative detection kit for HLA-B5801 alleles, which comprises the primer and probe combination.
Specifically, the kit also contains a positive quality control product and a negative quality control product; the positive quality control product is a plasmid containing the detected HLA-B5801 allele fragment and the detected internal standard gene fragment; the negative quality control product is purified water.
Specifically, the kit also contains reagents required by PCR reaction.
Specifically, reagents required for the PCR reaction include PCR Buffer, dNTPs, DNA polymerase, UDG enzyme and enzyme diluent.
Specifically, the PCR Buffer consists of (NH 4 ) 2 SO 4 、Tris-HCl、MgCl 2 Tween-20 and water.
Specifically, the DNA polymerase is a hot start Taq abzyme.
The invention also provides a method for qualitatively detecting HLA-B5801 allele, or a using method of the kit, which comprises the following steps:
s1, extracting nucleic acid of a sample to be detected;
s2, respectively taking the nucleic acid, the positive quality control product and the negative quality control product obtained by extracting in the step S1 as templates, preparing a PCR reaction system by using the primer and probe combination, and carrying out PCR amplification to obtain Ct values of different detection channels in each reaction system;
s3, judging a result;
negative quality control: FAM, texas-Red and CY5 detection channels have no obvious exponential growth period, and have no Ct value or Ct value >35;
controlling the nature of yang: FAM, texas-Red, CY5 detection channel has obvious logarithmic amplification curve and Ct value is less than or equal to 33;
sample to be detected: the internal standard CY5 channel has an obvious logarithmic amplification curve, ct is less than or equal to 35, the condition is met, the HLA-B5801 allele FAM detection channel has an obvious logarithmic amplification curve, ct value is less than or equal to 35, and the Ct value is less than or equal to 6 with the delta Ct value of the internal standard CY5 channel, the detection result is 5801 positive, namely the HLA-B5801 allele is carried; the internal standard CY5 channel has an obvious logarithmic amplification curve, ct is less than or equal to 35, the condition is met, the HLA-B5801 allele FAM detection channel has an obvious logarithmic amplification curve, ct value is less than or equal to 35 and delta Ct value of the internal standard CY5 channel is more than 6, and the detection result is 5801 negative, namely the HLA-B5801 allele is not carried; in addition, when the detection result of the FAM detection channel is underwined, Δct is recorded as "/", and is regarded as Δct >6;
the above requirements are met simultaneously in the same experiment, otherwise, the experiment is ineffective, and it is recommended to confirm the quality of the nucleic acid or sample and then re-detect.
Optionally, the sample is human whole blood.
Specifically, in the PCR reaction system described in the step S2, the concentration range of the primers 5801-F and 5801-R is 0.24 to 0.4 pmol/. Mu.L, the concentration range of the probe 5801-P is 0.12 to 0.2 pmol/. Mu.L, the concentration range of the internal standard probe is 0.15 to 0.25 pmol/. Mu.L, the concentration range of the internal standard probe is 0.08 to 0.16 pmol/. Mu.L, the concentration range of the hot start Taq antibody enzyme is 0.08 to 0.2U/. Mu.L, the concentration range of the UDG enzyme is 0.01 to 0.03U/. Mu.L, and the concentration range of dNTPs (A: G: C: U=1:1: 1: 2) is 0.4 to 0.5 nmol/. Mu..
Preferably, in the PCR reaction system described in step S2, the concentration of the primers 5801-F and 5801-R is 0.32 pmol/. Mu.L, the concentration of the probe 5801-P is 0.16 pmol/. Mu.L, the concentration of the internal standard probe is 0.2 pmol/. Mu.L, the concentration of the internal standard probe is 0.1 pmol/. Mu.L, the concentration of the hot start Taq abzyme is 0.1U/. Mu.L, the concentration of the UDG enzyme is in the range of 0.02U/. Mu.L, and the concentration of dNTPs (A: G: C: U=1:1: 1:2) is 0.4 nmol/. Mu.L. In contrast, the PCR reaction system has the best detection effect under the condition of the concentration.
Specifically, the fluorescence channel is set as: 1) The PCR reaction tube selects the FAM channel (Reporter: FAM, quantiser: none), CY5 channel was selected (Reporter: CY5, quantier: none) detects an internal standard channel; 2) The reference fluorescence (Passive Reference) was selected to be None.
Specifically, the PCR reaction conditions were 50℃for 2min;95 ℃ for 15min;94℃for 15s,55℃for 45s,40 cycles and fluorescence collected at this stage; the volume of the reaction system was set to 25. Mu.L.
The invention has the following beneficial effects:
the invention provides a primer and probe combination capable of detecting HLA-B5801 allele based on a double-start oligonucleotide primer and a fluorescent PCR technology aiming at HLA-B5801 allele. On the basis, the invention provides a qualitative detection kit and a detection method for HLA-B5801 alleles by matching with reagents required by PCR reactions such as hot-start DNA polymerase and the like. By using the primer and probe combination provided by the invention, HLA-B5801 can be specifically amplified, other highly homologous alleles are not amplified, and HLA-B5801 alleles can be accurately detected.
Compared with the similar products at present, the kit and the method have the advantages of short detection time and low cost, the detection specificity of the primer and probe combination obtained by adjustment and optimization is strong, the kit and the method can be completely distinguished from B-1501 and other genes with high homology, can greatly reduce cross reaction, achieve higher specificity, can be used for rapid qualitative detection of HLA-B5801 alleles, and is beneficial to personalized medicine application of allopurinol.
Drawings
FIG. 1 shows the amplification curve of a positive sample of human HLA-B5801 allele, with ΔCt.ltoreq.6.
FIG. 2 shows the amplification curve of human HLA-B5801 allele negative samples, with ΔCt >6.
FIG. 3 shows the amplification curve of human HLA-B5801 allele negative samples, with ΔCt >6.
Note that: the blue amplification curve represents the "FAM" channel of interest and the yellow amplification curve represents the "CY5" internal standard channel.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 design of primer and Probe combinations and kit therefor
The invention designs a plurality of pairs of primers and a plurality of probes based on double-start oligonucleotide primers (dual priming oligonucleotide, DPO) and fluorescence PCR by taking a nucleotide sequence of HLA-B5801 allele as a template, and obtains a group of primer and probe combinations with high specificity and sensitivity for detecting the HLA-B5801 allele through continuous testing, adjustment and optimization. The nucleotide sequences of the primer and probe combinations are shown in Table 1:
TABLE 1 primer and probe combinations for detection of HLA-B5801 allele
| Sequence numbering | Primer/probe name | Nucleic acid sequence (5 '-3') |
| SEQ ID NO.1 | 5801-F | CAGCGACGCCGCGAGIIIIIGGACGGA |
| SEQ ID NO.2 | 5801-R | TGTAGTAGCGGAGCGCGA |
| SEQ ID NO.3 | GAPDH-DF2 | GATATTGTTGCCATCAATGACC |
| SEQ ID NO.4 | GAPDH-DR2 | GGGAATACGTGAGGGTATGAA |
| SEQ ID NO.5 | 5801-P | ACGGGGAGACACGGAACATGAAG |
| SEQ ID NO.6 | GAPDH-DP2 | CCTCCCACACCAGCTTTGG |
The primer 5801-F contains IIIII in addition to the base A/T/C/G, wherein IIIII is 5 hypoxanthine or deoxyhypoxanthine. In addition, the probe is also marked with different fluorescent groups; specifically, the 5 'end of probe 5801-P is marked with FAM, and the 3' end is marked with BHQ1; the probe GAPDH-DP2 is labeled with CY5 at the 5 'end and BHQ2 at the 3' end.
Based on the primer and probe combination, the invention also provides a qualitative detection kit for HLA-B5801 alleles. The kit contains a B5801 PCR reaction liquid A, B5801 PCR reaction liquid B, a positive quality control product and a negative quality control product which are used for preparing a PCR reaction system; the B5801 PCR reaction solution A contains a primer and probe mixed solution prepared by combining B5801 PCR Buffer with the primer and probe shown in the table 1; the B5801 PCR reaction liquid B contains dNTPs (containing dUTP), hot start Taq abzyme, UDG enzyme and hot start Taq enzyme diluent; the positive quality control product contains the detected HLA-B5801 gene fragment and the detected HLA-B5801 gene fragmentPlasmids of internal standard Gene (GAPDH) fragments; the negative quality control product is purified water; the main component in the B5801 PCR Buffer is (NH) 4 ) 2 SO 4 、Tris-HCl、MgCl 2 And Tween-20.
Taking 25 mu L of reaction system as an example, 17 mu L B X5801 PCR reaction liquid A, 3 mu L B X5801 PCR reaction liquid B and 5 mu L of sample nucleic acid are added during preparation. The concentrations of each component in the PCR reaction solution and the optimal amounts thereof are shown in table 2, so that the optimal concentrations of each component in the PCR reaction system can be obtained, and the components are mixed in proportion to prepare the B5801 PCR reaction solution a and the B5801 PCR reaction solution B.
TABLE 2 optimal PCR reaction System
Example 2 qualitative detection of HLA-B5801 allele
Based on the kit described in example 1, the present invention also provides a qualitative detection method of HLA-B5801 allele, or a method of using the kit, comprising the steps of:
1. human whole blood sample collection and nucleic acid extraction
At least 1mL of venous blood of a subject is extracted and injected into an EDTA or sodium citrate-containing anticoagulation blood collection tube, and the glass tube is gently inverted and mixed for 5-10 times, so that the anticoagulation agent and the venous blood are fully and uniformly mixed, and the sealing and the preservation are carried out; extracting sample nucleic acid; the nucleic acid extraction or purification reagent used in the invention is a Guangdong ear mechanical equipment 20170666 reagent, and specific operation steps are shown in a reagent instruction book.
2. PCR detection
(1) Preparing a PCR reaction system
Taking out the B5801 PCR reaction liquid A and the B5801 PCR reaction liquid B from the kit, placing the mixture at room temperature, melting, shaking and mixing the mixture uniformly, and centrifuging the mixture at 8,000rpm for a plurality of seconds for use; n (N=number of samples to be tested+B+5801 positive quality control product+negative quality control product) PCR reaction tubes are taken, the total amount of each reaction tube to be prepared is calculated according to the dosage, and mixed solutions are prepared respectively.
(2) Placing the PCR reaction tube into a sample groove of an instrument, recording the placing sequence and selecting a fluorescent channel; setting negative quality control (NTC), 5801 positive quality control and Unknown sample (Unknown) according to the corresponding sequence of the samples, and setting sample names; the fluorescence channel is set as: 1) The PCR reaction tube selects the FAM channel (Reporter: FAM, quantiser: none), CY5 channel was selected (Reporter: CY5, quantier: none) detects an internal standard channel; 2) The reference fluorescence (Passive Reference) was selected to be None.
(3) The PCR conditions shown in Table 3 were set, and the volume of the reaction system was set to 25. Mu.L.
TABLE 3PCR reaction conditions
(4) Analyzing and judging results;
after the reaction is finished, automatically storing the result, regulating the Start Value, end Value and Threshold Value of Baserine according to the analyzed image (a user can adjust the Start Value at 3-15, the End Value can be set at 5-20 according to the actual situation, and the Value of Threshold in a Log map window is set to be 1/10 of the highest fluorescence Value of 5801 positive quality control, so that a Threshold line is positioned in an exponential phase of an amplification curve, the amplification curve of a negative quality control is straight or lower than the Threshold line), and clicking Analysis to automatically obtain the Analysis result. In the result analysis, the Ct value of each channel can be seen in the View Well Table, wherein the target detection signal is Ct (FAM), and the internal standard signal is Ct (CY 5).
And (3) result judgment:
negative quality control: FAM, texas-Red and CY5 detection channels have no obvious exponential growth period, and have no Ct value or Ct value >35;
controlling the nature of yang: FAM, texas-Red, CY5 detection channel has obvious logarithmic amplification curve and Ct value is less than or equal to 33;
sample to be detected: the internal standard CY5 channel has an obvious logarithmic amplification curve, ct is less than or equal to 35, the condition is met, the HLA-B5801 allele FAM detection channel has an obvious logarithmic amplification curve, ct value is less than or equal to 35, and the Ct value is less than or equal to 6 with the delta Ct value of the internal standard CY5 channel, the detection result is 5801 positive, namely the HLA-B5801 allele is carried; the internal standard CY5 channel has an obvious logarithmic amplification curve, ct is less than or equal to 35, the condition is met, the HLA-B5801 allele FAM detection channel has an obvious logarithmic amplification curve, ct value is less than or equal to 35 and delta Ct value of the internal standard CY5 channel is more than 6, and the detection result is 5801 negative, namely the HLA-B5801 allele is not carried; in addition, when the detection result of the FAM detection channel is unesterimed, Δct is recorded as "/", regarded as Δct >6;
the above requirements are met simultaneously in the same experiment, otherwise, the experiment is ineffective, and it is recommended to confirm the quality of the nucleic acid or sample and then re-detect.
The invention uses 1 case of clear HLA-B5801 positive and 2 cases of HLA-B5801 negative sample nucleic acid as template, uses the kit and the method to detect, the amplification curves of positive samples and 2 cases of negative samples are shown in figures 1-3 in turn, and the corresponding Ct values are shown in table 4.
Table 4 Ct values for HLA-B5801 positive and HLA-B5801 negative samples
| Sample of | Ct(CY5) | Ct(FAM) | △Ct |
| Positive sample | 24.374 | 26.485 | 2.111 |
| Negative sample 1 | 24.352 | Undetermined | / |
| Negative sample 2 | 22.924 | 34.446 | 11.522 |
Note that: when the FAM channel is "uncovered", Δct is recorded as "/", considered as Δct >6.
As can be seen from the results shown in fig. 1 to 3 and table 4, the kit of the present invention can specifically detect HLA-B5801 allele positive samples, and can be used for detection of HLA-B5801 allele.
Example 3 negative and Positive coincidence rate detection
The results of 8 nucleic acids containing other HLA-B alleles (i.e., HLA-B5801 negative clinical samples, numbered N1-N8) and non-human genomic nucleic acids (E.coli nucleic acids and Staphylococcus aureus nucleic acids, numbered N9-N10) were tested using the kit described in example 1 and the method described in example 2 and are shown in Table 5. As shown in table 5, the detection results were all HLA-B5801 negative, and the coincidence rate was 100%.
TABLE 5 negative coincidence rate detection results
| Numbering device | Detection result | Numbering device | Detection result |
| N1 | HLA-B5801 negative | N6 | HLA-B5801 negative |
| N2 | HLA-B5801 negative | N7 | HLA-B5801 negative |
| N3 | HLA-B5801 negative | N8 | HLA-B5801 negative |
| N4 | HLA-B5801 negative | N9 | HLA-B5801 negative |
| N5 | HLA-B5801 negative | N10 | HLA-B5801 negative |
The invention uses the kit described in example 1, and detects 10 parts of HLA-B5801 enterprise positive reference (with the numbers of P1-P10, wherein P1-P8 are HLA-B5801 positive clinical samples, P9 are HLA-B5801 positive cells, P10 is HLA-B5801 positive plasmid) and 8 parts of other allelic enterprise reference (the reference is a cell strain, the types are determined by the third party HLA parting service company through sequencing, the numbers are J1-J8, the types are HLA-B1402/4001, B5101/5201, B4101/5101, B0701/5101, B0702/0702, B3501/3901, B5101/45003 and B5108/8, and the genome of one cell is divided by 2 genes, as shown in table 5106). As shown in Table 6, the detection results all match the corresponding types, and the positive match rate was 100%.
TABLE 6 detection results of Enterprise Positive reference
Example 4 detection Limit and specificity detection
The standardized 0.5 ng/. Mu.L reference with the lowest detection limit of the enterprise in the detection range of the kit is detected, the detection is repeated for 3 times, the detection results are shown in the table 7, and the results accord with HLA-B5801 gene positivity. As shown in Table 7, the detection limit of the kit of the invention is 0.5 ng/. Mu.L, the Ct value range of the CY5 channel is 28-32, the Ct value range of the FAM channel is 31-35, and the detection rate is 100%, which indicates that the detection sensitivity is high and the repeatability is good.
TABLE 7 minimum limit of detection results
| Numbering device | Detection result |
| L1 | HLA-B5801 positive |
| L1 | HLA-B5801 positive |
| L1 | HLA-B5801 positive |
| L2 | HLA-B5801 positive |
| L2 | HLA-B5801 positive |
| L2 | HLA-B5801 positive |
| L3 | HLA-B5801 positive |
| L3 | HLA-B5801 positive |
| L3 | HLA-B5801 positive |
| L4 | HLA-B5801 positive |
| L4 | HLA-B5801 positive |
| L4 | HLA-B5801 positive |
| L5 | HLA-B5801 positive |
| L5 | HLA-B5801 positive |
| L5 | HLA-B5801 positive |
According to the results in the table 7, the positive rate of the detection result of the accuracy of each quality control product is 100%, which shows that the accuracy detection of the kit meets the requirements.
In addition, to examine the specificity of the primer and probe combinations shown in Table 1 of the present invention, plasmids containing other HLA-B alleles including HLA-B5701, B5802 alleles susceptible to cross-reaction, and HLA-B alleles having a frequency of more than 3% in Chinese population, including HLA-B1301, B1302, B1502, B4001, B4006, B4601, B5101 and B5401, were examined by the method described in example 2 using the kit described in example 1. As shown in Table 8, the results of specific detection are shown in Table 8, and the primer and probe composition of the present invention were negative for all the above-mentioned other alleles homologous to HLA-B5801, and it was found that there was no cross reaction with the above-mentioned alleles, indicating high detection specificity.
TABLE 8 specificity test results
| Sample of | Detection result |
| B5701 plasmid | HLA-B5801 negative |
| Plasmid B5802 | HLA-B5801 negative |
| Plasmid B1301 | HLA-B5801 negative |
| B.1302 plasmid | HLA-B5801 negative |
| B.1502 plasmid | HLA-B5801 negative |
| B4001 plasmid | HLA-B5801 negative |
| B4006 plasmid | HLA-B5801 negative |
| B4601 plasmid | HLA-B5801 negative |
| B5101 plasmid | HLA-B5801 negative |
| Plasmid B5401 | HLA-B5801 negative |
According to the results in the table, the coincidence rate of the detection result of the specificity of each plasmid sample is 100%, which shows that the specificity detection of the kit meets the requirements.
Example 5 clinical blood sample nucleic acid detection
In order to further verify the effect of the kit in actual detection, 1mL of 150 human peripheral whole blood is extracted and placed in blood collection tubes (numbered 1-150) of EDTA anticoagulant, and after the blood sample is extracted by nucleic acid, sequencing and typing are carried out by a gold standard PCR-SBT method, and meanwhile, the kit is used for detecting the blood sample. As can be seen from the results of the gold standard PCR-SBT method, samples numbered 4, 5, 17, 18, 19, 21, 31, 33, 41, 43, 48, 56, 58, 62, 63, 65, 74, 77, 80, 82, 103, 114, 116, 129, 133 and 136 were HLA-B5801 positive, carrying the HLA-B5801 gene, and none of the remaining numbered samples carried the HLA-B5801 allele. The results of the kit of the invention for detecting 150 clinical blood sample nucleic acids are shown in Table 9, and the results obtained by the kit of the invention completely accord with the parting results of gold standard as shown in Table 9.
TABLE 9 nucleic acid test results of 150 clinical blood samples
Comparative example
The invention designs a plurality of pairs of primers and probes aiming at HLA-B5801 allele, but detection tests show that the detection effects of different primers and probes and combinations thereof are obviously different.
For example, the primer and probe combinations of the present invention shown in Table 10 as a comparison, in which a nonspecific curve was found to appear in the specific assay, could not accurately distinguish between other alleles with high homology to HLA-B5801, such as HLA-B5701, B5702, B5703, etc. In contrast, the primer and probe combinations shown in Table 11, which are comparative, show an improvement in detection specificity, but have a large Ct value of the amplification curve when detecting a low concentration (0.5 ng/. Mu.L) of nucleic acid sample, resulting in failure to obtain an effective amplification curve when detecting a low concentration or lower. The detection method is described in example 2.
Table 10 control primer and Probe 1
TABLE 11 control primer and Probe 2
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A primer and probe combination for qualitatively detecting HLA-B5801 allele, comprising primer 5801-F/R and probe 5801-P; the nucleotide sequence of the primer 5801-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer 5801-R is shown as SEQ ID NO.2, and the nucleotide sequence of the probe 5801-P is shown as SEQ ID NO. 5.
2. The primer and probe combination of claim 1, further comprising an internal standard primer GAPDH-DF2/DR2 and an internal standard probe GAPDH-DP2; the nucleotide sequence of the internal reference primer GAPDH-DF2 is shown as SEQ ID NO.3, the nucleotide sequence of the internal reference primer GAPDH-DR2 is shown as SEQ ID NO.4, and the nucleotide sequence of the internal reference probe GAPDH-DP2 is shown as SEQ ID NO. 6.
3. The primer and probe combination of claim 2, wherein the probe 5801-P is labeled FAM at the 5 'end and BHQ1 at the 3' end; the probe GAPDH-DP2 is labeled with CY5 at the 5 'end and BHQ2 at the 3' end.
4. Use of a primer and probe combination according to any one of claims 1-3 for the preparation of a qualitative detection product for HLA-B5801 allele.
5. A qualitative detection kit for HLA-B5801 allele, comprising a primer and probe combination according to any one of claims 1 to 3.
6. The kit according to claim 5, wherein the kit further comprises a positive quality control and a negative quality control; the positive quality control product is a plasmid containing the detected HLA-B5801 allele fragment and the detected internal standard gene fragment; the negative quality control product is purified water.
7. The kit according to claim 5, wherein the kit further comprises reagents required for a PCR reaction.
8. The kit of claim 7, wherein the reagents required for the PCR reaction comprise PCR Buffer, dNTPs, DNA polymerase, UDG enzyme and enzyme diluent.
9. The kit of claim 8, wherein the PCR Buffer consists of (NH 4 ) 2 SO 4 、Tris-HCl、MgCl 2 Tween-20 and water.
10. The kit of claim 8, wherein the DNA polymerase is a hot start Taq abzyme.
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| CN202310749748.0A CN117187374A (en) | 2023-06-21 | 2023-06-21 | Qualitative detection kit for HLA-B5801 allele |
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