CN113862342A

CN113862342A - Primer group, probe, kit and method for detecting HLA-DR13 gene

Info

Publication number: CN113862342A
Application number: CN202111398643.2A
Authority: CN
Inventors: 林祥华; 林泽曦
Original assignee: Xiamen Abbot Medical Technology Co ltd
Current assignee: Xiamen Abbot Medical Technology Co ltd
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2021-12-31

Abstract

The application discloses a primer group, a probe, a kit and a method for detecting HLA-DR13 gene, which belongs to the technical field of biomedical engineering, wherein the primer group for detecting HLA-DR13 gene and the corresponding probe thereof comprise a primer group and a probe designed according to HLA-DR13 allele specificity, and the primer group comprises nucleotide sequences shown as SEQ ID NO: 1 and the forward primer shown as SEQ ID NO: 2, and a reverse primer; the nucleotide sequence of the probe is shown as SEQ ID NO: 3 is shown in the specification; or a complement thereof. The application has the effects of shortening the time for detecting the HLA-DR13 gene, improving the detection efficiency and carrying out high-throughput detection on the HLA-DR13 gene.

Description

Primer group, probe, kit and method for detecting HLA-DR13 gene

Technical Field

The application relates to the technical field of biomedical engineering, in particular to a primer group, a probe, a kit and a method for detecting HLA-DR13 gene.

Background

Human Leukocyte Antigen (HLA) is an expression product of the human Major Histocompatibility Complex (MHC), is distributed on leukocyte surfaces and systemic histiocytes, determines the histocompatibility of the body, and is regulated and expressed by a group of genes on human chromosome 6. The chemical nature of HLA is a kind of glycoprotein, which is formed by non-covalent combination of a glycosylated alpha heavy chain and a beta light chain, wherein the amino terminal of the peptide chain faces outwards, the carboxyl terminal penetrates into cytoplasm, and the middle hydrophobic part is positioned in the cell membrane.

HLA, a genetic marker of human tissue cells, particularly HLA on immune cells, has biological properties of transferring antigen polymorphisms to antigen-specific T Cell Receptors (TCRs), and plays an important role in antigen recognition, immune response, immune regulation, and the like. HLA is a genetic system which is found in human bodies for the first time and has a clear relationship with diseases, is the most complex human gene complex known at present, and can be divided into I, II and III genes according to functions.

Among them, class ii antigens are ligands of CD4 molecules, and are involved in processes such as T cell responses, genetic control of immune responses, restriction of immune cell-cell interactions, antigen presentation, immunomodulation, and immune cell differentiation. The HLA-DRB polymorphism is most complex in known HLA class II antigens, more than 227 alleles have been found in the gene coding regions of the antigens, human immune response genes are positioned in the gene coding regions of the antigens, and the strength of the human immune response is controlled by the HLA-DR genes. In recent studies, the HLA-DR gene is found to be closely related to the pathogenesis process of chronic severe hepatitis B.

The chronic hepatitis B refers to a person with over half a year of positive Hepatitis B Virus (HBV) detection, or with uncertain disease onset date and clinical chronic hepatitis manifestation, and the clinical manifestations of the chronic hepatitis are symptoms such as hypodynamia, anorexia, nausea, liver pain and the like; among them, chronic severe hepatitis b is a common fatal cause of hepatitis b.

After a human body is infected by HBV, an immune system is activated to generate an immune response reaction, but the immune system attacks own liver cells due to the excessively strong immune response in an organism, so that a large amount of liver cells are necrotized and apoptotic, and liver failure is caused; in the process, the internal environment of the body is changed, so that the intestinal mucosa barrier function is reduced, the liver Kupffer cell phagocytosis function is reduced, the endotoxin in the body is increased, the Kupffer cell is activated to release substances such as cell factors and inflammatory transmitters, the hepatic circulation disorder is caused, the liver is attacked secondarily, the hepatic injury is continuously existed and is continuously aggravated, and finally the chronic severe hepatitis B is caused.

It is considered that the progression of the disease course after infection of a human body with HBV depends mainly on the immune response status of the individual, and virus-specific T cell responses play a key role in the clearance of HBV viruses, especially HLA-DR-restricted specific CD4+ T cells, which induce the development of HBV virus-specific Cytotoxic T Lymphocyte (CTL) responses, and helper B lymphocytes produce antigens against the HVB outer membrane and nucleocapsid. Wherein, HLA-DR13 can present more HBV antigenic determinants than other HLA-DR, thereby inducing the body to generate stronger HVB virus specific CD4+ T cell reaction, leading the body immune response to be over-strong to cause a great deal of necrosis and apoptosis of liver histiocytes, and leading the generation of chronic severe hepatitis B. The HLA-DR13 gene has close relation with the basic method or the possible severe hepatitis of the chronic hepatitis B, and is a valuable index for judging the disease condition and predicting the course of the disease.

Currently, the HLA-DR13 gene detection method mainly comprises a Sanger sequencing method, which utilizes the principle that the 3' end of ddNTP does not contain hydroxyl and can not form phosphodiester bond to cause the interruption of DNA synthesis. In the Sanger sequencing process, a DNA polymerase is used to extend primers bound to a specific sequence template, and after synthesis has proceeded to some extent, a fluorescently labeled ddNTP is added to the reaction system to generate a plurality of sets of nucleotides of different lengths ending with different nucleotides, which are then electrophoresed on a urea-denatured PAGE gel for detection. The Sanger sequencing method has higher accuracy in detecting the HLA-DR13 gene, but the Sanger sequencing method is more complicated to operate, requires longer time and is not suitable for high-flux detection.

Disclosure of Invention

In order to shorten the time for detecting the HLA-DR13 gene and improve the detection efficiency, the application provides a primer group, a probe, a kit and a method for detecting the HLA-DR13 gene, wherein the primer group, the probe and the kit are used for carrying out high-throughput detection on the HLA-DR13 gene.

In a first aspect, the present application provides a primer set and a corresponding probe thereof for detecting HLA-DR13 gene, which adopts the following technical scheme:

a primer group and a corresponding probe for detecting HLA-DR13 gene comprise a primer group and a probe which are designed according to the specificity of HLA-DR13 allele, wherein the primer group comprises nucleotide sequences shown as SEQ ID NO: 1 and the forward primer shown as SEQ ID NO: 1, a reverse primer; the nucleotide sequence of the probe is shown as SEQ ID NO: 1 is shown in the specification; or a complement thereof.

By adopting the technical scheme, the primer group and the probe are designed according to the specificity of the HLA-DR13 allele, the forward primer and the reverse primer can be respectively and tightly base-complementarily paired with two single strands of a DNA chain containing the HLA-DR13 allele to realize effective specific amplification of the HLA-DR13 gene sequence, the probe can identify the specific site on the HLA-DR13 gene and be base-complementarily paired with the specific site, and the specific detection of the HLA-DR13 gene is realized through the two base-complementary pairings of the primer group, the probe and the HLA-DR13 gene.

In a second aspect, the present application provides a kit, which adopts the following technical scheme:

a kit comprises the primer group and the probe for detecting the HLA-DR13 gene.

Preferably, the kit further comprises an internal standard primer and an internal standard probe.

Preferably, the internal standard primer group and the internal standard probe are designed according to a conserved sequence on an HLA-II gene.

Preferably, the internal standard primer group comprises nucleotide sequences shown as SEQ ID NO: 1 and a forward internal standard primer shown as SEQ ID NO: 1, reverse internal standard primer; the nucleotide sequence of the internal standard probe is shown as SEQ ID NO: 1 is shown.

By adopting the above technical scheme, the conserved sequence refers to a molecular sequence with high similarity or identity, which exists on all HLA genes; in the process of detecting the HLA-DR13 gene, the internal standard primer and the internal standard probe which are specifically designed by the conserved sequence are used as reference objects, so that the operation error in the sample loading process can be corrected, the effectiveness of a detection system is ensured, and the accuracy of detecting the HLA-DR13 gene is improved.

Preferably, the probe and the internal standard probe are fluorescent probes.

Preferably, the probe and the internal standard probe are labeled with fluorescent groups, and the fluorescent groups are independently selected from FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, VIC, HEX or ROX.

Preferably, the probe and the internal standard probe are labeled with a quenching group, and the quenching group is independently selected from BHQ-0, BHQ-1, BHQ-2, Dabcyl, Eclipse or TAMRA.

By adopting the technical scheme, the two ends of the probe and the internal standard probe are respectively marked with the fluorescent probe and the quenching group, and the quenching group absorbs fluorescence emitted by the fluorescent group; when detection is carried out, the probe or the internal standard probe is specifically combined with a DNA single strand in a DNA sample to be detected and participates in the synthesis of the DNA, the fluorescent group and the quenching group are hydrolyzed from the probe and the internal standard probe in the process of DNA amplification, so that the fluorescent group and the quenching group are separated, the fluorescent group emits fluorescence to be detected by a detection system to detect signals, and whether the HLA-DR13 gene exists in the detection system can be qualitatively analyzed by detecting whether the fluorescent signal exists in the amplification system.

Preferably, the kit further comprises a PCR reaction system, wherein the PCR reaction system comprises 10 xTris-HCl buffer solution, soluble magnesium salt, dNTP and Taq DNA polymerase.

In a third aspect, the present application provides a method for detecting HLA-DR13 gene, which adopts the following technical scheme:

a method for detecting HLA-DR13 gene, using the kit to perform real-time fluorescence PCR.

By adopting the technical scheme, the reagent in the kit and the DNA sample to be detected are uniformly mixed and put into a fluorescence PCR instrument for amplification, and the qualitative judgment on whether the HLA-DR13 gene exists in the DNA sample to be detected can be realized by detecting whether a fluorescence signal exists in an amplification system, the detection method is to detect the amplification product while amplifying, the operation steps are simple, the detection time is only 2 hours, the detection time is effectively shortened, and the rapid detection on the HLA-DR13 gene is further realized.

In summary, the present application includes at least one of the following beneficial technical effects:

(1) the application provides a primer group and a probe for real-time fluorescence PCR detection of HLA-DR13 gene, wherein a forward primer and a reverse primer in the primer group can be specifically combined with the HLA-DR13 gene, if a DNA sample to be detected contains the HLA-DR13 gene, specific effective amplification can be carried out, the probe is also specifically combined with the HLA-DR13 gene, in the process of synthesis and extension, a fluorescent group and a quenching group on the probe are hydrolyzed by Taq DNA synthetase, so that the fluorescent group and the quenching group are separated, whether the HLA-DR13 gene exists in the DNA sample to be detected can be qualitatively analyzed by judging whether a fluorescent signal exists in an amplification system, an amplification product is detected while amplification is carried out, the process of detecting the HLA-DR13 gene is simplified, and the detection time is shortened, the detection efficiency is improved, and the high-throughput detection of the HLA-DR13 gene can be realized.

(2) The primer and the probe in the application are low in price, sequencing is not needed in the detection process, the detection cost is saved, the detection period is shortened, and the detection efficiency is improved.

Drawings

FIG. 1 is a photograph of agarose gel electrophoresis in example 1.

Detailed Description

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 # R0191, Thermo Fisher Co.).

The present application is described in further detail below with reference to fig. 1.

The real-time fluorescent PCR technology is a method for detecting whether polymerase chain reaction occurs in an amplification system in real time by using a fluorescent signal in a DNA amplification reaction, and comprises a Taqman probe method and a Sybrgreen I dye method. Wherein, a probe capable of emitting fluorescence is added in the process of PCR amplification in the Taqman probe method to detect a specific product amplified and synthesized during the circulation, the fluorescent probe can be specifically combined with a target sequence, the 5 'end and the 3' end are respectively modified with a fluorescent group and a quenching group, when the probe and the target sequence are combined and participate in polymerase chain reaction, the fluorescent group and the quenching group are hydrolyzed from the probe by Taq DNA polymerase, so that the fluorescent group and the quenching group are separated, and a fluorescent signal is detected in an amplification system. The Taqman probe method generates a fluorescent signal through specific hybridization between the fluorescent probe and a target, does not need to carry out electrophoresis on an amplification product after the amplification is finished, directly realizes the detection of the amplification product through the detection of the fluorescent signal in the amplification process, and has the advantages of high specificity, reduction of analysis workload, detection cost saving and the like.

According to the application, a group of primer groups and probes are obtained by designing primer design software according to the nucleotide sequence of HLA-DR13 allele, the primer groups and the probes are used for carrying out real-time fluorescence PCR detection on a DNA sample known to contain the HLA-DR13 gene, a fluorescence signal is not detected in a system during amplification, the used primer groups and probes are not suitable to cause PCR amplification failure, so that a plurality of groups of primer groups and probes are manually designed, the primer groups and the probes are used for carrying out real-time fluorescence PCR detection, and finally a group of primer groups and probes with good detection effect are determined, wherein the nucleotide sequences of the primer groups and the probes are shown as follows:

a forward primer: SEQ ID NO: 1;

reverse primer: SEQ ID NO: 2;

and (3) probe: SEQ ID NO: 3.

in order to correct operation errors in the sample loading process in the fluorescent PCR detection and ensure the effectiveness of a detection system, an internal standard primer group and an internal standard probe are introduced into a real-time fluorescent PCR detection system. The internal standard primer group and the internal standard probe are designed according to the specificity of a conserved sequence on an HLA-D region gene, and the nucleotide sequences are as follows:

forward internal standard primer: SEQ ID NO: 4;

reverse internal standard primer: SEQ ID NO: 5;

internal standard probe: SEQ ID NO: 6.

the application provides a kit, which comprises the primer group and the probe, and an internal standard primer group and an internal standard probe, and is used for performing real-time fluorescence PCR detection on HLA-DR13 genes. Wherein, the probe and the internal standard probe are fluorescent probes, the 5 'end of the probe and the internal standard probe are marked with fluorescent groups, and the 3' end of the probe and the internal standard probe are marked with quenching groups; the fluorophore can be one of FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, VIC, HEX, and ROX, and the quencher can be one of BHQ-0, BHQ-1, BHQ-2, Dabcyl, Eclipse, and TAMRA.

The kit provided by the application also comprises a PCR reaction system, and the reagent composition of the reaction system is shown in Table 1:

TABLE 1

Reagent	Dosage of
		10 × Tris-HCl buffer	2.5μL
dNTP	0.06mM each
		Magnesium chloride	0.3mM
Potassium chloride	20.1mM
		Glycerol	0.2％
Taq DNA polymerase	0.2mM
		ddH₂O	Adding to 25 μ L

The application also provides a method for detecting the human HLA-DR13 gene, the method uses the kit to carry out real-time fluorescence PCR detection on a DNA sample to be detected, and detects an amplification product in the PCR amplification process, so that the amplification product does not need to be subjected to electrophoresis after the amplification is finished, only about 1h of detection time is needed, the detection time is effectively shortened, the detection efficiency is improved, and the high-throughput detection of the HLA-DR13 gene can be realized.

Example 1: primer group and probe for detecting HLA-DR13 gene

The design of primers and probes is an important step in gene detection, and currently, design software is usually used to design primer sets and probes according to the specificity of target sequences to be detected. In this example, the inventors designed a set of primer sets and probes based on the nucleotide sequence of the HLA-DR13 gene by designing software, and the nucleotide sequences of the primer sets and probes are as follows:

a forward primer: SEQ ID NO: 7;

reverse primer: SEQ ID NO: 8;

and (3) probe: SEQ ID NO: 9.

synthesizing the primer group by a chemical synthesis company to obtain a synthesis test report, performing real-time fluorescent PCR detection on a DNA sample known to contain HLA-DR13 gene by using the primer group and the probe, adding an internal standard primer group and an internal standard probe into a detection system, modifying a fluorescent group on the 5 'end of the probe to be VIC, modifying a fluorescent group on the 5' end of the internal standard probe to be FAM, and collecting fluorescent signals through different signal paths; it was found that the fluorescence signal of the fluorophore labeled on the internal standard probe could be detected in the amplification system, but the fluorescence signal of the fluorophore labeled on the probe could not be detected, indicating that the specific amplification of HLA-DR13 gene did not occur in the amplification system, and the amplification failed.

Therefore, the inventors manually designed 6 primer sets based on the nucleotide sequence of HLA-DR13 gene, and the nucleotide sequence is shown in Table 2.

TABLE 2

And 5 sets of the primer sets and the probes were synthesized by chemical synthesis, and then, a DNA sample known to contain HLA-DR13 gene was subjected to PCR amplification using the 5 sets of the primer sets and the probes, respectively, and the resulting amplification product was subjected to agarose gel electrophoresis.

The results obtained are shown in FIG. 1, where 1 to 5 correspond to primer sets No. 1 to 5, respectively, and amplified bands appear in 1, 2 and 4: the band in 1 appeared at 137bp, the band in 2 appeared at 178bp, the band in 4 appeared at 296bp, and the amplified bands in 3 and 5 did not appear; PCR is carried out by using the

primer groups

1, 2 and 4 to obtain an amplification product, the

primer groups

1 and 2 obtain a specific amplification product, and the primer group 4 does not obtain a specific amplification product; it is demonstrated that specific amplification of HLA-DR13 can be achieved by using primer sets No. 1 and No. 2, while specific amplification of HLA-DR13 cannot be achieved by primer sets No. 3, No. 4 and No. 5, and specific amplification effect of primer set No. 2 is the best.

The inventors designed 5 probes based on the nucleotide sequence of the amplification product specifically using primer set No. 2, and the nucleotide sequences of the probes are shown in Table 3:

TABLE 3

Numbering	Nucleotide sequence
			1	SEQ ID NO：18
2	SEQ ID NO：19
		3	SEQ ID NO：20
4	SEQ ID NO：3
		5	SEQ ID NO：21

The results of real-time fluorescence PCR of DNA samples known to contain HLA-DR × 13 gene were performed using the 5 probe sets and the 2 primer set, respectively, and a blank control set was set, which used DNA samples known to not contain HLA-DR × 13 gene, and the results are shown in table 4:

TABLE 4

As can be seen from table 4: no. 1 and No. 3 probes are used in real-time fluorescent PCR amplification, and fluorescent signals cannot be detected, so that the two groups of probes have problems and cannot perform specific detection on HLA-DR13 genes; the No. 5 probe can also detect a fluorescent signal in a blank control group, which indicates that the probe has no specificity; the real-time fluorescent PCR using the No. 2 and No. 4 probes can detect fluorescent signals in a reaction system, but the Ct value of the No. 2 probe is large, the quality of the probe is poor, and the detection result is greatly influenced.

Therefore, in this example, primer set 2 and probe 4 were used as the primer set and probe for HLA-DR13 gene detection.

Example 2: method for detecting HLA-DR13 gene

1. Extraction of DNA sample to be tested

In the detection of HLA-DR13 gene, the gene can be derived from tissue cellsOr extracting the DNA sample to be detected from the plasma. In this example, a 15min high-yield whole blood genomic DNA extraction kit was used to extract a DNA sample to be tested from a blood sample of a subject, and the concentration and purity of the extracted DNA sample to be tested were determined using a GeneQuant Pro nucleic acid protein concentration determinator, at a concentration of greater than 10 ng/. mu.L and an OD of OD_260nm/OD_280nmThe value of (A) is between 1.7 and 2.0.

2. PCR amplification primer set and probe

Primer pairs and probe sets for detection of HLA-DR13 gene were synthesized by the synthesis company and a synthesis test qualification report was provided. The nucleotide sequences of the primer set and the probe are as follows:

a forward primer: SEQ ID NO: 1;

reverse primer: SEQ ID NO: 2;

and (3) probe: SEQ ID NO: 3.

in this example, an internal standard primer set and an internal standard probe were added to a real-time fluorescent PCR detection system to check the effectiveness of the detection system. And the fluorescent group marked at the 5 'end of the probe is VIC, and the fluorescent group marked at the 5' end of the internal standard probe is FAM.

3. Real-time fluorescent PCR reaction

In this example, a kit containing a PCR reaction system, a primer set, a probe, an internal standard primer set, and an internal standard probe was used to perform a real-time fluorescent PCR reaction; the reagents and the amounts of the reagents in the PCR reaction system are shown in Table 5:

TABLE 5

Reagent	Concentration/amount
		10 × Tris-HCl buffer	2.5μL
dNTP	0.06mM each
		Forward primer/reverse primer	0.6. mu.L each
Taq DNA polymerase	0.2mM
		Magnesium chloride	0.3mM
Potassium chloride	20.1mM
		Glycerol	0.2％
DNA sample to be tested	2μL
		ddH₂O	Adding to 25 μ L

The reagents in the table 5 are added into a centrifuge tube for 120rmp, centrifuged and mixed uniformly for 30s, then the mixture is placed into a fluorescence PCR instrument for real-time fluorescence PCR detection according to the cycle program shown in the table 6, and a positive control and a negative control are arranged at the same time of detection.

TABLE 6

And in the step of annealing extension, the fluorescence signal acquisition is carried out on the amplification system, and the signal acquisition is carried out by using a double channel of FAM (670nm) and VIC (560 nm).

4. Analysis of results

In this example, 4 whole blood samples were subjected to HLA-DR x 13 gene assay, and the assay results are shown in table 7:

TABLE 7

From Table 7, it was found that the whole blood samples No. 1 and No. 3 were negative for HLA-DR13 gene, while the samples No. 2 and No. 4 were positive for HLA-DR13 gene.

Example 3: specific assay for the method of detecting HLA-DR13 gene 7 samples of whole blood known not to contain HLA-DR13 gene and 1 sample of whole blood known to contain HLA-DR13 gene were assayed using the method of detecting HLA-DR13 gene described in example 2.

Firstly, 8 parts of the whole blood sample is extracted by using a 15min high-yield whole blood genome DNA extraction kit to obtain a DNA sample, and then the extracted DNA sample is subjected to real-time fluorescence PCR detection, and the obtained results are shown in Table 8:

TABLE 8

As can be seen from table 8: the detection result of the positive control substance is positive, and the detection result of the negative control substance is negative; among the 8 whole blood samples, the test results of the whole blood samples No. 1-7 were negative, and the test results of the whole blood sample No. 8 were positive, which indicates that the specificity of the method for detecting HLA-DR13 gene described in example 2 is high, as the known conditions are known.

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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Claims

1. A primer group and a corresponding probe for detecting HLA-DR13 gene are characterized by comprising a primer group and a probe which are designed according to the specificity of HLA-DR13 allele, wherein the primer group comprises nucleotide sequences shown as SEQ ID NO: 1 and the forward primer shown as SEQ ID NO: 2, and a reverse primer; the nucleotide sequence of the probe is shown as SEQ ID NO: 3 is shown in the specification; or a complement thereof.

2. A kit comprising the primer set for detecting HLA-DR13 gene and the probe of claim 1.

3. The kit of claim 2, further comprising an internal standard primer and an internal standard probe.

4. The kit according to claim 3, wherein the internal standard primer group and the internal standard probe are designed based on a conserved sequence on HLA-D region genes.

5. The kit according to claim 4, wherein the internal standard primer set comprises a nucleotide sequence shown as SEQ ID NO: 4 and a forward internal standard primer shown as SEQ ID NO: 5, reverse internal standard primer; the nucleotide sequence of the internal standard probe is shown as SEQ ID NO: and 6.

6. The kit of claim 5, wherein the probe and internal standard probe are fluorescent probes.

7. The kit of claim 6, wherein the probe and the internal standard probe are labeled with fluorophores independently selected from FAM, TET, JOE, HEX, CY3, TAMRA, ROX, Texas Red, VIC, HEX, and ROX.

8. The kit of claim 7, wherein said probe and internal standard probe are labeled with a quencher group, said quencher group is independently selected from the group consisting of BHQ-0, BHQ-1, BHQ-2, Dabcyl, Eclipse and TAMRA.

9. The kit of claim 8, further comprising a PCR reaction system comprising 10 × Tris-HCl buffer, soluble magnesium salt, dntps, Taq DNA polymerase.

10. A method for the detection of HLA-DR13 gene, characterized in that real-time fluorescent PCR is performed using the kit of claim 9.