CN113981051A - Primer group, kit and method for detecting HLA-DPB 0202 gene - Google Patents
Primer group, kit and method for detecting HLA-DPB 0202 gene Download PDFInfo
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Abstract
The application discloses a primer group, a kit and a method for detecting HLA-DPB 0202 genes, which belong to the technical field of biomedical engineering, wherein the primer group for detecting HLA-DPB 0202 genes comprises a forward outer primer F3, a forward inner primer FIP, a backward outer primer B3 and a backward inner primer BIP; the nucleotide sequence of the forward outer primer F3 is shown as SEQ ID NO. 2; the nucleotide sequence of the forward inner primer FIP is shown as SEQ ID NO. 3; the nucleotide sequence of the backward outer primer B3 is shown as SEQ ID NO. 4; the nucleotide sequence of the backward inner primer BIP is shown as SEQ ID NO. 5. The kit has the effects of rapidly detecting the HLA-DBP1 x 0202 gene and shortening the detection time.
Description
Technical Field
The application relates to the technical field of biomedical engineering, in particular to a primer group, a kit and a method for detecting HLA-DPB 0202 gene.
Background
Graves' disease (GD), also known as toxic diffuse goiter, is an immunological disease of autoimmune disorders whose clinical manifestations are not limited to the thyroid gland but rather a multisystem syndrome including hypermetabolic syndrome, diffuse goiter, ocular signs, skin lesions and thyroacropathy; and GD is seen in families with cases of subsequent onset, which are closely linked to genetic genes, scientists speculate that GD may be a disease regulated by Human Leukocyte Antigen (HLA) genes. HLA is the expression product of the Major Histocompatibility Complex (MHC) in humansThe substance, with 224 loci and more than 2000 alleles, has been found to be the most complex group of genes in humans to date, which can be classified into class i and class ii antigens based on distribution and function. The II antigen is mainly distributed on the surfaces of antigen presenting cells, thymic epithelial cells and activated T cells, is coded by an HLA-D region on a chromosome 6, comprises HLA-DPB, HLA-DQB, HLA-DRB and other types, and is heterodimer glycoprotein formed by non-covalently combining glycosylated alpha heavy chain and beta light chain. Based on the location of class II antigens, they can be divided into extracellular, transmembrane and intracellular regions; the extracellular region can be further divided into a peptide binding region and a polypeptide region, and the antigen polymorphism of the II antigen depends on a beta 1 structural domain on the polypeptide region. The peptide binding region and the polymorphic region of the class II antigen are cooperatively combined to present exogenous antigen peptide to CD4+T cells having a function of regulating T cell differentiation.
According to research, abnormal autoimmune response appears in GD patients, thyroid stimulating immunoglobulin (TSAb) is secreted by B cells, and the TSAb is combined with thyroid follicular cells to induce the uncontrolled secretion of thyroid hormone, so that inflammatory cells infiltrate into thyroid tissues to cause the generation of thyroid inflammation; meanwhile, the patient also secretes inhibitory thyroid stimulating hormone binding immunoglobulin (TBII), and the TBII plays a negative feedback regulation role in the secretion of TSAb from B cells. Thus, TSAb and TBII positivity were detected in general GD patients, but in recent studies, it was found that patients with genes such as HLA-A0201, HLA-A0207 and HLA-DPB 0202 were negative for TBII in their blood, and patients who were negative for TBII were more effective in clinical treatment than those who were positive, and GD was classified into typicals and atypical based on TBII, and GD classification was achieved by detection of genes such as HLA-A0201, HLA-A0207 and HLA-DPB 0202.
Gene amplification is one of basic research means in the field of molecular biology, and the traditional Polymerase Chain Reaction (PCR) and the technologies such as Sanger sequencing method, fluorescence PCR and the like developed on the basis can realize effective amplification and detection of biological nucleic acid.
Currently, Sanger sequencing is generally used for the detection of HLA-DPB 0202 gene, and the Sanger sequencing utilizes the principle that chain extension is terminated due to the lack of 3' -OH required for PCR extension by dideoxynucleoside triphosphate (ddNTP). However, the Sanger sequencing method obtains multiple DNA fragments by performing multiple amplifications, and requires electrophoresis of the amplified DNA, which is cumbersome to operate and requires a long time for detection.
Disclosure of Invention
In order to rapidly detect the HLA-DBP 0202 gene and shorten the detection time, the application provides a primer group, a kit and a method for detecting the HLA-DPB 0202 gene.
In a first aspect, the present application provides a primer set for loop-mediated isothermal amplification (LAMP) detection of HLA-DPB 0202 genes, which adopts the following technical scheme:
a primer group for detecting HLA-DPB 0202 genes comprises a forward outer primer F3, a forward inner primer FIP, a backward outer primer B3 and a backward inner primer BIP;
the nucleotide sequence of the forward outer primer F3 is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward inner primer FIP is shown as SEQ ID NO. 3;
the nucleotide sequence of the backward outer primer B3 is shown as SEQ ID NO. 4;
the nucleotide sequence of the backward inner primer BIP is shown as SEQ ID NO. 5.
In a second aspect, the kit provided by the present application adopts the following technical scheme:
a kit comprising the primer group for detecting the HLA-DPB 0202 gene.
Preferably, dNTP, Bst DNA polymerase, betaine and nuclease-free water are also included.
By adopting the technical scheme, when detection is carried out, a kit containing a primer group, dNTP, Bst DNA polymerase, betaine and nuclease-free water is used, and if a sample to be detected contains HLA-DPB 0202 genes, specific amplification of the HLA-DPB 0202 genes can be realized; and the kit contains four primers, so that the amplification specificity of the HLA-DPB 0202 gene is stronger, and the detection accuracy is improved.
Preferably, an indicator is also included.
Preferably, the indicator is a soluble magnesium salt.
By adopting the technical scheme, when the kit is used for detection, if a sample contains HLA-DPB 0202 genes, an amplification reaction can be carried out, a byproduct pyrophosphate ion can be generated in the amplification reaction process, the pyrophosphate ion can react with soluble magnesium salts in the kit to generate magnesium pyrophosphate precipitate, and whether the white precipitate is generated or not can be judged, so that whether the sample to be detected contains the HLA-DPB 0202 genes or not can be judged without other instruments, the detection operation is simplified, and the detection efficiency is improved.
Preferably, the indicator is a dye.
Preferably, the dyes are independently selected from hydroxynaphthol blue indicating dyes and calcein indicating dyes.
By adopting the technical scheme, when an amplification product exists in an amplification system, a dye has a specific color reaction, and whether a sample to be detected contains the HLA-DPB 0202 gene can be judged by judging the color of the system, so that the judgment of a detection result is facilitated; when the dye is a hydroxy naphthol blue indicating dye and a sample to be detected contains HLA-DPB 0202 genes, the reaction system is subjected to specific amplification and is sky blue, otherwise, the reaction system is violet; when the dye is calcein indicating dye and the sample to be detected is positive by HLA-DPB 0202, the reaction system is subjected to specific amplification and is green, otherwise, the reaction system is orange.
Preferably, a positive control and a negative control are also included.
Preferably, the positive control is an artificially synthesized DNA fragment containing HLA-DPB 0202 allele, the nucleotide sequence of the DNA fragment is shown as SEQ ID NO:1, and the negative control is nuclease-free water.
By adopting the technical scheme, the positive control substance and the negative control substance are added into the kit, the situation that the detection result is wrong due to false negative can be eliminated by the positive control substance containing the DNA fragment of the artificially synthesized HLA-DPB 0202 allele, the negative control substance is used for judging whether the amplification system is polluted to cause false positive or not without nuclease water, and the accuracy of the detection of the HLA-DPB 0202 gene can be improved by matching the positive control substance and the negative control substance.
In a third aspect, the method for detecting HLA-DPB 0202 genes provided by the present application employs the following technical scheme: a method for detecting HLA-DPB 0202 gene, comprising the step of subjecting any of the above kits to LAMP amplification.
In summary, the present application includes at least one of the following beneficial technical effects:
(1) the kit provided by the application comprises a primer group for detecting HLA-DPB 0202 genes, dNTP, Bst DNA polymerase, betaine, nuclease-free water and an indicator, wherein the reagent provided by the kit is an amplification reaction system of loop-mediated isothermal amplification technology (LAMP), and the detection of the HLA-DPB 0202 genes is realized through the amplification reaction system; if the sample to be detected contains the HLA-DPB 0202 gene, LAMP amplification reaction occurs, the LAMP amplification can be performed only under the condition of constant temperature, the amplification is completed for about 60min, and the detection result can be interpreted through the indicator, so that the effects of simplifying detection operation and effectively shortening detection time are realized, the detection efficiency is improved, no expensive instrument is required in the detection process, and the detection cost is saved;
(2) the positive control substance and the negative control substance are added into the kit, so that false positive results and false negative results can be eliminated, and the reliability of the HLA-DPB 0202 gene detection result is improved.
Drawings
FIG. 1 is a schematic diagram of the LAMP amplification initiator synthesis stage;
FIG. 2 is a schematic diagram of the amplification cycle stage of LAMP amplification;
FIG. 3 is an agarose gel electrophoresis of LAMP amplification products of the five primer sets in example 1;
FIG. 4 shows the result of the HLA-DPB 0202 gene assay of a whole blood sample according to example 2;
FIG. 5 shows the results of specific detection by the method for detecting HLA-DPB 0202 gene in example 3;
fig. 6 shows the results of the sensitivity test in the method for detecting HLA-DPB 0202 gene in example 4.
Detailed Description
The present application is described in further detail below with reference to figures 1-6.
The reagent or kit and sources thereof referred to in this application are as follows:
DNA ladder (cat 11721933001, Merck);
15min high-yield whole blood genome DNA extraction kit (product number B0136, Haerbin New Haishi Gene detection Co., Ltd.);
dNTP Mix (Cat. No. R0191, Thermo Fisher Co., Ltd.);
bst DNA polymerase (product number 12908ES60, Saint Biotech (Shanghai) Co., Ltd.);
betaine (Cat. B802386-100g, Macklin Co.);
calcein indicating dye (cat # 40719ES50, san Jose Biotech (Shanghai) Co., Ltd.).
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, and a method for efficiently and quickly completing an amplification reaction under an isothermal condition by using DNA polymerase with strand displacement activity and two pairs of specially designed primers. Compared with the traditional amplification detection method, the LAMP technology overcomes the defect that the traditional PCR reaction needs to obtain a single-chain template through repeated thermal denaturation, avoids the process of repeated temperature rise and fall, realizes continuous and rapid amplification under the constant temperature condition, and has higher sensitivity and amplification efficiency; and the LAMP can not carry out nucleic acid amplification if the sequence to be detected can not be completely matched with the six regions by aiming at the four specific primers designed by the LAMP for the six regions of the target sequence, so that the LAMP technology has stronger specificity.
LAMP technology, which is proposed by Notomi et al, has a core of nucleic acid amplification consisting of a DNA polymerase having strand displacement activity and two pairs of specific primers. BstDNA polymerase is an enzyme with strand displacement activity and can be used as an enzyme for catalyzing DNA amplification in LAMP technology; the two pairs of primers with strong specificity comprise a pair of inner primers and a pair of outer primers, wherein the inner primers comprise a forward inner primer FIP and a backward inner primer BIP, and the outer primers comprise a forward outer primer F3 and a backward outer primer B3.
FIG. 1 is a schematic diagram of the LAMP amplification initiator synthesis stage: four primers FIP, F3, BIP and B3 are designed aiming at six different sites of F3c, F2c and F1c at the 3 'end and B1, B2 and B3 at the 5' end, any primer is subjected to base pairing extension to the complementary part of the double-stranded DNA so as to release the double-stranded DNA, F2 on FIP is combined with the complementary sequence F2c of the template, under the action of BstDNA polymerase, the DNA synthesis is started from the 3 'end of F2, a new DNA single-strand with FIP as the starting point is synthesized and is combined with the template strand to form the double-stranded DNA, then the new strand with F3 as the starting point is combined with the template strand to form the double-stranded DNA, the originally synthesized DNA single-strand with FIP as the starting point is replaced to generate a single-stranded DNA, F1c and F1 regions on the 5' end of the DNA are subjected to base pairing to form a stem-loop structure, and simultaneously the B2 of BIP is combined with the template strand c as the new template strand with the initial point, and complementary with the template strand to form a DNA double strand, and finally, the B1c and B1 regions at the 5' end of the DNA double strand undergo base complementary pairing to form a stem-loop structure, and the whole strand is dumbbell-shaped.
FIG. 2 is a schematic diagram of the LAMP amplification cycle stage: taking a DNA double strand with a stem-loop structure as a template, combining FIP with an F2c region of a stem loop, and carrying out strand displacement synthesis to dissociate the DNA double strand with the stem-loop structure to form single-stranded nucleic acid which also forms a ring structure; meanwhile, taking the B1 segment at the 3' end as a starting point, taking the B1 segment as a template, carrying out DNA synthesis extension and strand displacement to form two new stem-loop structure DNAs with different lengths, carrying out complementary pairing on the B2 on the BIP and the base thereof, starting a new round of amplification, obtaining DNA mixtures with different number of stem-loop structures and different lengths at the stage, and taking the amplified DNA as an alternate inverted repeat sequence of a target sequence.
LAMP amplification comprises two stages of initiator synthesis and cyclic amplification: a stem circular DNA is finally formed in the synthesis stage of the starting material and is used as a template in the cyclic amplification stage; in the cyclic amplification stage, a displacement reaction is carried out under the catalysis of the inner primer and BstDNA polymerase, stem-loop structures with different lengths are formed under the combined action of the outer primer, and the final amplification product is a mixture consisting of stem-loop DNA and cauliflower-like DNA.
Designing a plurality of primer groups according to six different regions on the HLA-DPB 0202 allele with the nucleotide sequence shown as SEQ ID NO. 1, synthesizing the primer groups by a chemical synthesis company, and performing LAMP amplification on DNA samples containing the HLA-DPB 0202 allele by using the five primer groups respectively to obtain poor effect; therefore, the nucleotide sequence of the primer group is adjusted to realize the modification of the primer, and finally a group of primer groups with good LAMP amplification effect is obtained.
In the present application, the adjusted primer set was used as a primer set for HLA-DPB x 0202 gene detection, and the nucleotide sequence of the primer set was as follows:
forward outer primer F3: 2, SEQ ID NO;
forward inner primer FIP: 3, SEQ ID NO;
rear-outward primer B3: 4, SEQ ID NO;
backward inner primer BIP: SEQ ID NO 5.
The application also provides a kit, and the kit contains the primer group, reagents required by the LAMP amplification reaction system and an indicator for indicating the detection result. When the detection is carried out, the reagent in the kit and the DNA sample to be detected only need to be uniformly mixed, the reaction is carried out for about one hour under the condition of constant temperature of 60 ℃, and the detection result is judged by the indicator. Compared with the most common Sanger sequencing method, the detection time of the method only needs one to two hours, the detection time is effectively shortened, and the detection efficiency of the HLA-DPB 0202 gene is improved.
In the kit provided by the present application, the reagent composition of the LAMP amplification reaction system is shown in table 1.
TABLE 1
When any primer carries out base complementary pairing extension to a complementary part of the double-stranded DNA, the double-stranded DNA is opened and dissociated into two single strands, an F2 sequence in FIP is combined with a template F2c, Bst DNA polymerase catalyzes a strand displacement synthesis reaction, and four dNTPs are utilized to synthesize a new single-stranded DNA; and the betaine has the function of stabilizing Bst DNA polymerase, and is beneficial to the LAMP amplification reaction.
In addition, a byproduct pyrophosphate is generated in the nucleic acid amplification process, and the occurrence of LAMP amplification reaction is judged by detecting whether the pyrophosphate is generated in the reaction system, so that the target gene is judged whether exists in the reaction system. At present, methods for isothermal amplification of nucleic acids using pyrophosphate mainly include a turbidity method and a colorimetric method.
Also included in the kits provided herein are indicators for pyrophosphate, including both soluble magnesium salts and dyes.
Wherein the soluble magnesium salt is MgCl2And MgSO4Adding a soluble magnesium salt into a reaction system before LAMP amplification, wherein the soluble magnesium salt is dissolved in water and exists in an ion form; when LAMP amplification occurs, magnesium ions in the reaction system react with pyrophosphate to generate magnesium pyrophosphate precipitate, and the occurrence of LAMP amplification is judged according to whether the precipitate is generated in the reaction system, so that the detection of whether HLA-DPB 0202 gene exists in a DNA sample to be detected is realized, and the effect of easily judging the detection result is realized.
The dye used to indicate pyrophosphate is one of a hydroxynaphthol blue indicating dye and a calcein indicating dye. The hydroxyl naphthol blue indicating dye comprises Hydroxyl Naphthol Blue (HNB) and magnesium ions, a complex formed by combining HNB and the magnesium ions is violet, when LAMP amplification occurs in a system to generate pyrophosphate, the pyrophosphate reacts with the magnesium ions to generate magnesium pyrophosphate precipitate, the HNB loses the magnesium ions to make the color of the system be sky blue, and the detection result can be judged through the change of the color of the system; the principle of the calcein indicating dye discoloration indicating detection result is similar to that of HNB indicating dye: when LAMP amplification does not occur, a compound formed by combining calcein and manganese ions is orange yellow, if pyrophosphate is generated by LAMP amplification, the pyrophosphate reacts with the manganese ions to generate manganese pyrophosphate precipitate, and the calcein loses the manganese ions to make the system green.
In addition, the indicator may be a dye directly labeling the amplified and synthesized DNA, and may be independently selected from one of SYBR Green I, Ethidium Bromide (EB) and CFI fluorescent dyes.
In addition, in order to improve the reliability of the detection result of the HLA-DPB 0202 gene, the kit provided by the application also comprises a positive control substance and a negative control substance. The positive control product can be a standard DNA sample which is obtained by separation and purification and contains the HLA-DPB 0202 allele fragment, and can also be a DNA fragment which is artificially synthesized and contains the HLA-DPB 0202 allele; the negative control can be a negative DNA sample which is obtained by separation and purification and is known not to contain HLA-DPB 0202 allele fragments, and can also be nuclease-free water, wherein the nucleotide sequence of the HLA-DPB 0202 allele is shown as SEQ ID NO. 1.
LAMP amplification of a positive control and a negative control is carried out under the same reaction system and reaction conditions, and if the LAMP amplification occurs to the positive control, the detection system provided by the kit is proved to be effective, so that the influence on the detection result caused by the phenomenon of false negative of LAMP amplification is reduced; if the LAMP amplification does not occur in the negative control, the influence of false positive phenomenon caused by non-specific amplification caused by primer dimer on the detection result can be eliminated; by arranging the positive reference substance and the negative reference substance, the influence on the judgment of the detection result caused by false positive and false negative phenomena can be reduced, and the effect of improving the accuracy of the detection result is realized.
Example 1: primer group for detecting HLA-DPB 0202 gene
Five primer sets for LAMP amplification were designed from HLA-DPB x 0202 allele by PrimerExplorer V5 website, and the nucleotide sequences of the five primer sets are shown in Table 2. The five primer sets were synthesized by chemical synthesis, and each primer set was LAMP-amplified with respect to a DNA sample known to contain HLA-DPB x 0202 gene at a concentration of 10 ng/. mu.L and nuclease-free water, and the product obtained by LAMP amplification was subjected to agarose gel electrophoresis.
TABLE 2
The obtained electrophoresis results are shown in FIG. 3, in which the 1-2 wells are the 1 st group primer set, the 3-4 wells are the 2 nd group primer set, the 5-6 wells are the 3 rd group primer set, the 7-8 wells are the 4 th group primer set, and the 9-10 wells are the 5 th group primer set; from the electrophoresis results, it was found that the 1 st and 5 th primer sets showed bands at positions close to 200bp, LAMP amplification did not occur in the 3 rd and fourth primer sets, and false positive occurred in the 2 nd primer set.
From the results, it was found that specific LAMP amplification occurred only in the 1 st and 5 th primer groups among the five primer groups, but the LAMP amplification effect was not good in the two primer groups. Therefore, on the basis of the first group and the fifth group of primer groups, the primer groups are modified by changing the nucleotide sequences of the primers to obtain a plurality of groups of primer groups, and the primer groups are used for respectively carrying out LAMP amplification on a DNA sample known to contain HLA-DPB 0202 gene and nuclease-free water and carrying out electrophoresis on LAMP amplification products, so that a group of primer groups with the best LAMP amplification effect is finally ensured, wherein the nucleotide sequences of the primer groups are shown as follows:
forward outer primer F3: 2, SEQ ID NO;
forward inner primer FIP: 3, SEQ ID NO;
rear-outward primer B3: 4, SEQ ID NO;
backward inner primer BIP: SEQ ID NO 5.
Example 2: method for detecting HLA-DPB 0202 gene
1. Extraction of DNA from sample to be tested
In this example, the presence or absence of HLA-DPB 0202 gene in a whole blood sample was detected, and DNA was extracted from the whole blood sample using a DNA extraction kit, and the DNA extraction was performed according to the instructions provided in the DNA extraction kit.
LAMP amplification primer set A primer set for LAMP detection of HLA-DPB 0202 gene was synthesized by a synthetic company and provided a synthesis test qualification report, and the nucleotide sequences of the primer sets were as follows:
forward outer primer F3: 2, SEQ ID NO;
forward inner primer FIP: 3, SEQ ID NO;
rear-outward primer B3: 4, SEQ ID NO;
backward inner primer BIP: SEQ ID NO 5.
LAMP amplification reaction
In this example, a kit containing dNTP, Bst DNA polymerase, betaine, nuclease-free water, calcein indicator dye, a positive control, and a negative control was used for LAMP amplification reaction. When a whole blood sample is detected, 3 LAMP reaction tubes are needed, namely a negative control tube, a positive control tube and a detection tube, and the reaction system of each LAMP reaction tube is shown in Table 3.
TABLE 3
| Reagent | Dosage of |
| 10×buffer | 2.5μL |
| FIP/ |
2 μ L each |
| F3/B3 | 0.5. mu.L each |
| dNTP Mix | 3μL |
| Bst DNA polymerase | 1μL |
| Betaine | 4μL |
| Calcein indicating dye | 2.5μL |
| DNA sample/negative control | 2μL |
| Nuclease-free water | 5μL |
Before amplification, the reagents except the DNA sample and the negative control in the table 2 are melted at room temperature and centrifuged at 2000rmp for 10s to be uniformly mixed, then the negative control, the positive control and the DNA sample to be detected are respectively added into a negative control tube, a positive control tube and a detection tube, and the reaction is carried out for 60min at the constant temperature of 62 ℃; after the reaction is finished, the LAMP reaction tube is cooled to room temperature, centrifuged at 2000rmp for 10s, and the color of the LAMP reaction tube is observed under an ultraviolet LAMP.
FIG. 4 shows the test result of a whole blood sample, which is a negative control tube, a positive control tube and a test tube from left to right; the negative control tube is green, the LAMP amplification does not occur, the positive control tube is orange, the LAMP amplification occurs in the negative control tube, and the effectiveness of the LAMP reaction system can be determined through the negative control tube and the positive control tube; and the detection tube is orange yellow, which indicates that the whole blood sample contains HLA-DPB 0202 gene.
Example 3: specificity detection of method for detecting HLA-DPB 0202 gene
Nine samples of whole blood known not to contain HLA-DPB 0202 genes and one sample of whole blood known to contain HLA-DPB 0202 genes were tested using the kit described in example 1.
Firstly, extracting the DNA of the ten whole blood samples by using a DNA extraction kit, then melting the reagents except the DNA sample and the negative control in the table 2 at room temperature, centrifuging at 2000rmp for 10s, uniformly mixing, then respectively adding the negative control, the positive control and the DNA sample to be detected into a negative control tube, a positive control tube and a detection tube, and reacting for 60min at the constant temperature of 62 ℃; after the reaction is finished, the LAMP reaction tube is cooled to room temperature, centrifuged at 2000rmp for 10s, and the color of the LAMP reaction tube is observed under an ultraviolet LAMP.
The results are shown in FIG. 5, where tubes Nos. 1-9 are green and tube No. 10 is orange. Tubes 1-9 are nine whole blood samples known to not contain the HLA-DPB 0202 gene, tubes 10 are whole blood samples known to contain the HLA-DPB 0202 gene, and the results show that only tube 10 is positive, while tubes 1-9 are negative, which indicates that only whole blood samples containing the HLA-DPB 0202 gene are positive, and the kit provided by the application has stronger specificity.
Example 4: sensitive detection of method for detecting HLA-DPB 0202 gene
HLA-DPB 0202 alleles with nucleotide sequences shown in SEQ ID NO. 1 are artificially synthesized, DNA samples of HLA-DPB 0202 alleles with a series of concentrations are prepared, and the sample concentrations and corresponding LAMP reaction tubes are shown in Table 4.
The obtained detection results are shown in fig. 6, and tubes 1-6 show positive results, while tubes 7-8 show negative results, and from the above results, it can be seen that the minimum detection limit for detecting HLA-DPB 0202 gene by LAMP technique is 0.5 ng/. mu.l, indicating that the method of this example has high detection sensitivity.
TABLE 4
| Numbering | Concentration (ng/. mu.L) |
| 1 | 100 |
| 2 | 50 |
| 3 | 10 |
| 4 | 5 |
| 5 | 1 |
| 6 | 0.5 |
| 7 | 0.1 |
| 8 | 0.01 |
The primer group, the kit and the method for detecting the HLA-DPB 0202 gene have the advantages of short detection time, high detection efficiency, strong specificity, high sensitivity and the like.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
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<213> Artificial Sequence (Artificial Sequence)
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gcgctgacgg gcgagga 17
<210> 12
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cccacgtcgc tgtcgaagca cgcgtttaat gggacacagc gc 42
<210> 13
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gggacggcag gaatgct 17
<210> 14
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ctgactgggg cggcgccagc tcgtatgtct ggttg 35
<210> 15
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtcgcgacgt cccagtag 18
<210> 16
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gatgccagtc cctttggctg tttacgtggg acacagcgcg tttaa 45
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
taccttttcc aggggcgac 19
<210> 18
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acctacacgg gagctcgtgg gacaggcttg gatgtcctt 39
<210> 19
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
acccggccgc ttgcc 15
<210> 20
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cgtcgctgtc gaagcgcgtt taagacacag cg 32
<210> 21
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gggacggcag gaatgct 17
<210> 22
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
tacatctaca accgggagga gctcgtgcca ggatgtcctt ctggctgtt 49
<210> 23
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tcctgtccgg cactg 15
<210> 24
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cccacgtcgc tgtcgaagct gggacacagc gcttcctg 38
<210> 25
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ccttttccag ggacggca 18
Claims (10)
1. A primer group for detecting HLA-DPB 0202 genes is characterized by comprising a forward outer primer F3, a forward inner primer FIP, a backward outer primer B3 and a backward inner primer BIP;
the nucleotide sequence of the forward outer primer F3 is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward inner primer FIP is shown as SEQ ID NO. 3;
the nucleotide sequence of the backward outer primer B3 is shown as SEQ ID NO. 4;
the nucleotide sequence of the backward inner primer BIP is shown as SEQ ID NO. 5.
2. A kit comprising the primer set for HLA-DPB 0202 gene detection according to claim 1.
3. The kit of claim 2, further comprising dntps, Bst DNA polymerase, betaine, and nuclease-free water.
4. The kit of claim 3, further comprising an indicator.
5. The kit of claim 4, wherein the indicator is a soluble magnesium salt.
6. The kit of claim 4, wherein the indicator is a dye.
7. The kit of claim 6, wherein the dyes are independently selected from hydroxynaphthol blue indicator dyes and calcein indicator dyes.
8. The kit of claim 3, further comprising a positive control and a negative control.
9. The kit according to claim 8, wherein the positive control is an artificially synthesized DNA fragment containing HLA-DPB 0202 allele, the nucleotide sequence of the DNA fragment is shown in SEQ ID NO. 1, and the negative control is nuclease-free water.
10. A method for detecting HLA-DPB 0202 gene, comprising the step of performing LAMP amplification using the kit of any one of claims 2 to 9.
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