CN111118150A

CN111118150A - Method, kit, primer pair and probe for detecting IL28B gene

Info

Publication number: CN111118150A
Application number: CN202010141524.8A
Authority: CN
Inventors: 李洪波; 张玉祥; 邢军芬; 朱滨
Original assignee: Shanghai Baio Technology Co ltd
Current assignee: Shanghai Baio Technology Co ltd
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2020-05-08
Anticipated expiration: 2040-03-02
Also published as: CN111118150B

Abstract

The invention discloses a method, a kit, a primer pair and a probe for detecting an IL28B gene. The kit comprises a PCR reaction system and a gene chip, wherein the PCR reaction system comprises a primer pair consisting of an upstream primer and a downstream primer, and the gene chip comprises a probe; the primer pair and the probe are selected from one or more combinations as described in the present invention. When the method, the kit, the primer pair and the probe are used for detecting the IL28B gene, the method is simple to operate, low in cost, high in sensitivity and easy to read and accurate in result. The method, the kit, the primer pair, the probe and the application thereof detect the IL28B genotype of a patient, actively predict the curative effect and the prognosis of the chronic hepatitis C patient on the Peg-IFN/RBV standard treatment scheme, guide a clinician to select and determine the drug variety and scheme at first and provide a simple and easy solution.

Description

Method, kit, primer pair and probe for detecting IL28B gene

Technical Field

The invention relates to the field of in-vitro diagnostic reagents, in particular to a method, a kit, a primer pair and a probe for detecting an IL28B gene.

Background

Blood is an important biological sample, and contains a large amount of DNA polymerase inhibitory substances such as heme, hetero-protein, fat, and the like. In order to complete the PCR amplification of nucleic acid from blood samples, the nucleic acid material must be purified from the blood. Generally, the method comprises three processes, namely, rupturing cell membranes and nuclear membranes; secondly, nucleic acid is purified, namely impurities such as protein and the like which influence the subsequent PCR reaction are removed; thirdly, eluting nucleic acid. Although nucleic acid with higher purity can be obtained through extraction treatment, the nucleic acid extraction process often results in a great amount of nucleic acid loss, and the complicated extraction and purification steps increase the risk of cross contamination among samples, thereby increasing the possibility of failure of subsequent PCR amplification. In addition, the extraction process is long in time consumption and high in cost, and some extraction methods also need phenol and other harmful reagents, so that the risk of infection of operators is increased, and high-throughput detection is difficult to realize. Therefore, the blood sample can be directly used as a template or simply treated to be used as a template for PCR amplification detection, which is a problem to be solved for blood sample amplification.

The gene for coding interleukin-28B (interleukin-28B, IL28B) is positioned on the 19 th chromosome (19q13.13) of a human body, consists of 6 exons and 5 introns, codes IFN-lambda 3, is a novel interleukin coding gene belonging to an IFN-lambda family, and has the full length of 1401 bp. rs12979860 is located 3kb upstream of the promoter of IL28B gene, rs8099917 is located 8.9kb upstream of IL28B gene, and rs12980275 is located 2.4kb downstream of the gene. In China, the CC type of the SNPrs12979860 locus of the IL28B gene accounts for 84.1 percent, the CT type accounts for 15.3 percent and the TT type accounts for 0.6 percent. Distribution of rs8099917 and rs12980275 is similar to SNP rs 12979860. The IL28B gene polymorphism has correlation with the virus infection chronization and Interferon (IFN) antiviral curative effect of hepatitis patients.

In recent years there have also been authoritative guidelines indicating the importance of IL28B for the treatment of hepatitis. In 2011, 3 months, the European liver research Association (EASL) introduced the "diagnosis and treatment guideline for HCV infection" at the head, and regarding the prediction of the efficacy of antiviral treatment, the EASL guideline increases the important prediction factor of the host genotype of IL28B, and the European liver disease society in 2014 updates the "diagnosis and treatment guideline for HCV infection". New researches also show that the IL28B gene polymorphism can also predict the triple therapeutic effect of Peg-IFN and ribavirin standard treatment and new small molecule drugs (DAA, such as telaprevin and the like) directly resisting HCV.

The american society for liver disease research (AASLD) updated the "guideline for treatment of chronic HCV infection genotype 1" 2014, which recommends that, for triple therapy with standard therapy in combination with DAA, the IL28B genotype is also a powerful factor in predicting a patient's sustained virological response, and that examination of the IL28B genotype of the patient should be considered prior to treatment.

On the 23 rd annual meeting of the asia-pacific liver research society (APASL) held by bris book of australia in 3 months 2014, the 2014 edition asia-pacific region "consensus on hepatitis c virus infection and treatment program" was published, and the status of IL28 gene in the treatment of hepatitis c was determined. The guidelines explicitly state that: the SNP at IL28B locus, rs12979860CC genotype, is a powerful determinant of HCV spontaneous clearance; acute hepatitis C can be observed for 8-16 weeks, awaiting spontaneous clearance, particularly in symptomatic patients. However, patients carrying the unfavorable IL28B gene phenotype can begin treatment as soon as 12 weeks ago because of the low likelihood of spontaneous clearance; because of the factor of IL28B, asian genotype 1 patients will require only 6 months of treatment with triple therapy. In addition, the FDA allows for the labeling of the IL28B gene in drug labels as genomic biomarkers for three drugs, peginteferon alfa-2b, Boceprevir, and Telaprevir. The current standard treatment regimen for hepatitis C is Peg-IFN/RBV.

Currently, many technical means such as fluorescent quantitative PCR, high-resolution melting curve, gene sequencing and the like are used for the detection of IL28B genotyping. The gene sequencing method is suitable for high-throughput multi-site detection, but is long in operation time and low in sensitivity, and is not suitable for rapid clinical detection; and the amount of information obtained by sequencing is large, much information is useless for patients, some professional knowledge is needed for judging the sequencing result, and the cost is relatively high. The high-resolution dissolution curve method has special requirements on equipment, can be used only by a machine which is provided with high-resolution software and sensitive to temperature, and has certain difficulty in clinical popularization.

Regarding gene sequencing method, CN105463084A discloses a method for detecting three SNP sites of human IL28B gene, comprising PCR amplification reaction reagent and PCR primer for amplifying three SNP sites of IL28B in sample DNA; collecting a blood sample to be detected, extracting DNA, carrying out PCR amplification by using the DNA as a template and PCR primers for amplifying three SNP sites of IL28B in the DNA of the sample to obtain a PCR reaction product, carrying out sequencing analysis, and determining the genotypes of rs12979860, rs8099917 and rs 12980275. The IL28B genotyping detection performed by the methods has the problems of high instrument purchase cost, need of extracting genome DNA, complex operation procedures and the like in different degrees, and is not beneficial to improving the detection efficiency and realizing clinical popularization. From the present, the detection requirement is huge, so that the search for a detection means which is efficient and easy to accept by patients has important practical significance for IL28B genotyping detection.

Furthermore, it is well known in the art that primer design takes into account the sequence composition (GC content) of the amplification product containing the SNP site, the size of the fragment, the position of the SNP site in the product, and the like, which are important factors affecting the hybridization kinetics of the PCR product with the probe, in genotyping. The size of a PCR product fragment with good hybridization kinetics is generally 200-300 bp, the steric hindrance of the PCR product with a larger fragment (for example, more than 500bp) is not favorable for hybridization with the probe, the hybridization efficiency is reduced, the hybridization efficiency of the PCR product with a smaller fragment (for example, less than 100bp) is higher, but the specific hybridization difficulty of wild and mutant probes is increased. The PCR product with higher GC content has higher hybridization efficiency due to stronger hydrogen bonding capability among bases, while the PCR product with lower GC content has lower efficiency under the same hybridization conditions. The position of the SNP site in the PCR product is also an important factor influencing the steric hindrance of hybridization. These factors make it not particularly easy to design primer pairs that can be used to genotype a particular SNP site. Similarly, the design of the probe needs to overcome many difficulties, such as ensuring that the hybridization conditions of the wild-type probe and the mutant-type probe are as consistent as possible. A great deal of experimental verification is needed to design better primer pairs and probes.

Disclosure of Invention

The invention aims to solve the technical problems that in the prior art, when genotyping is carried out, amplification efficiency is low, cost is high, an operation procedure is complex and time-consuming, popularization is not easy, cross contamination and toxic reagent use are increased, instability caused by multiple amplification and probe hybridization in the same amplification tube is high, cost is low, operation is simple, and popularization is easy, and the method, the kit, the primer pair, the probe and the application for detecting the IL28B gene are provided. The method has the advantages of good stability, low cost, simple operation and easy popularization and application. The method, the kit, the primer pair and the probe can be used for detecting the IL28B genotype, have high accuracy, can accurately distinguish various types of mutation sites, and can be used for guiding patients to take medicines. The gene chip in the kit has good signal-to-noise ratio, is not easily interfered by other interferents in samples to be detected, such as whole blood, oral exfoliative cells and the like, and solves the problem that amplification products of the samples to be detected, such as the whole blood, the oral exfoliative cells and the like, containing various interferents influence the hybridization of the chip. Wherein the probe and the primer pair have good specificity. The method and the kit have the advantages of simple operation and low cost, have high sensitivity (in a preferred embodiment of the invention, the detection limit can be as low as 900 white blood cells), greatly shorten the detection time, have easily-readable and accurate results, are very visual in judgment, and are more suitable for clinical detection and popularization. The method of the invention can preferably use the whole blood to-be-detected sample as the PCR template directly, and can avoid extracting the genome DNA, so that the method does not need to purchase a genome DNA extraction kit additionally, saves the cost, reduces the pollution chance, and is suitable for the detection of large-scale samples. And the required samples are few, and only 1 microliter of blood is needed at least (while 200 microliter of blood is generally needed in the prior art for DNA extraction), and the samples to be detected such as whole blood and the like are stored in a 4-degree refrigerator within 7 days, so that the detection result is stable, and reexamination is facilitated, or other samples to be detected such as whole blood and the like detected by a patient can be utilized, special sampling of the patient is not needed, and the pain of the patient is relieved. The invention can realize the detection of the IL28B gene by directly carrying out PCR hybridization on a sample to be detected. The hybridization step in the method can be realized through a full-automatic hybridization process, so that the method is more convenient and quicker and avoids a plurality of uncertain factors existing in the manual operation process. The method, the kit, the primer pair, the probe and the application thereof detect the IL28B genotype of a patient, actively predict the curative effect and the prognosis of the chronic hepatitis C patient on the Peg-IFN/RBV standard treatment scheme, guide a clinician to select and determine the drug variety and scheme at first and provide a simple and easy solution.

The existing method for genotyping generally adopts fluorescent quantitative PCR, even if strong-tolerance DNase is used, a sample to be detected can be used in the PCR only by certain pretreatment, and the requirement on the sample to be detected is high and the dosage is large. In addition, as known to those skilled in the art, in the environment of a sample to be tested (such as whole blood, exfoliated cells in the mouth, etc.) with an interfering substance, the design and the proportion of a primer pair and a probe are relatively difficult. In the hybridization reaction, the sequence composition, the lengths of the target DNA molecule and probe, the hybridization temperature, salts, etc. all affect the hybridization efficiency and strength. The amplification product used for genotyping cannot be too long, which would affect the hybridization speed, and when the amplification product is short, there is very little room for primer selection, while it is necessary to ensure sufficient amplification efficiency, and the design difficulty is very large in consideration of the cooperation with the primer. In addition, because the probe is fixed on the glass slide, and the glass slide has a certain repulsive force, the spatial structure of the probe needs to be considered when the probe is designed, so that the repulsive force is reduced; meanwhile, the designed probe is ensured to be capable of hybridizing with the amplification product; and the specificity of probe hybridization needs to be considered, and the homozygous mutant sample needs to ensure that no signal is generated when the wild-type probe is hybridized, and the mutant probe can generate a signal. The wild type and mutant type probes in the invention only have difference of a few bases, and the design difficulty is very large. The inventor of the invention has made a great deal of trials, and unexpectedly found that even if ordinary non-fluorescent quantitative PCR is used, PCR amplification and a gene chip are combined, and the primer pair and the probe of the invention are matched, the obtained detection result has high accuracy, sensitivity and specificity, the detection method is simple, rapid and low in cost, and samples such as whole blood, oral exfoliative cells and the like to be detected can be directly added for detection without any pretreatment.

In order to solve the above technical problems, the present invention aims to provide a kit for detecting IL28B gene, which comprises a PCR reaction system and a gene chip, wherein the PCR reaction system comprises a primer pair consisting of an upstream primer and a downstream primer, and the gene chip comprises a probe; wherein the content of the first and second substances,

the primer pair and the probe are selected from one or more of the following (1) to (3):

(1) the primer pair and the probe are used for detecting the rs12979860 locus of an IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 (5'-AGTCTGGGATTCCTGGACGTGG-3') and SEQ ID NO.2 (5'-GGCTCAGGGTCAATCACAGAAGGGAG-3'), and/or the nucleotide sequence of the probe is shown as SEQ ID NO.7 (5'-GAAGGCGCGAACCAGG-3') and/or SEQ ID NO.8 (5'-CGAAGGCGTGAACCAGG-3');

(2) the primer pair and the probe are used for detecting rs8099917 site of IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.3 (5'-CCCACTTCTGGAACAAATCG-3') and SEQ ID NO.4 (5'-TGGTATCAACCCCACCTCAA-3'), and/or the nucleotide sequence of the probe is shown as SEQ ID NO.9 (5'-TTCTGTGAGCAATTTCACCCAAATT-3') and/or SEQ ID NO.10 (5'-TCTGTGAGCAATGTCACCCAAAT-3');

(3) the primer pair and the probe are used for detecting the rs12980275 site of the IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.5 (5'-AGAGAGCAAGAGGAGGGAAG-3') and SEQ ID NO.6 (5'-CTGGTCCTAGTGGTGTTTGC-3'), and/or the nucleotide sequence of the probe is shown as SEQ ID NO.11 (5'-AATTCCTAGAAACAGACGTGTCTAA-3') and/or SEQ ID NO.12 (5'-TTCCTAGAAACGGACGTGTCTA-3').

In the present invention, the gene chip may be conventional in the art, and generally comprises a solid support and specific oligonucleotide probes immobilized on the solid support.

Preferably, the concentration of the primers in the PCR reaction system is 0.12-0.84. mu.M, preferably 0.2-0.72. mu.M, such as 0.14. mu.M.

More preferably, in the step (1), the concentration of the primer pair is 0.12-0.72. mu.M, and the concentration of the upstream primer is preferably 0.14. mu.M; and/or, the concentration of the downstream primer is preferably 0.6. mu.M.

More preferably, in the step (2), the concentration of the primer pair is 0.2-0.64. mu.M, and the concentration of the upstream primer is preferably 0.2. mu.M; and/or, the concentration of the downstream primer is preferably 0.6. mu.M.

More preferably, in the step (3), the concentration of the primer pair is 0.2-0.84. mu.M, and the concentration of the upstream primer is preferably 0.22. mu.M; and/or, the concentration of the downstream primer is preferably 0.84. mu.M.

Preferably, the concentration of the probe in the gene chip is 1.25-10. mu.M, such as 2.5. mu.M.

More preferably, the concentration of the probe for detecting the locus rs12979860 of the IL28B gene is 1.4-5 mu M, and preferably 3 mu M; and/or the concentration of the probe for detecting the rs8099917 site of the IL28B gene is 1.25-2.5 mu M, preferably 1.67 mu M; and/or the concentration of the probe for detecting the locus rs12980275 of the IL28B gene is 1.43-3.3 mu M, preferably 2.5 mu M.

Preferably, the primer pair is a primer pair modified by a biological detection marker at the 5' end, wherein the biological detection marker is preferably biotin, digoxin, fluorescein, a fluorescein derivative, a fluorescent molecule, alkaline phosphatase or horseradish peroxidase, and is more preferably biotin. By adopting the biological detection marker modification of different primer pairs, the corresponding detection method can be changed during the subsequent hybridization detection. These detection methods may be any known in the art.

In a preferred embodiment of the present invention, the primer pair is a primer pair with biotin modification at the 5' end, and the subsequent hybridization detection is also performed by using an anti-biotin antibody solution and a developing solution. The method specifically comprises the following steps: biotin (Biotin) on the amplification product first hybridized with the detection probe first reacts with Streptavidin-alkaline phosphatase complex (Streptavidin-AP) to form a new complex: Biotin-Streptavidin-AP, which undergoes a color reaction as follows:

Biotin-Stripavitin-AP+BCI-P→BCI-OH+Pi(pH 7.5)

BCI-OH + NBT → bluish purple precipitate

Wherein BCIP is 5 bromo-4 chloro-3 indolyl phosphate; NBT is nitro blue tetrazolium. The IL28B genotype was detected by hybridizing a probe to the amplified region.

Preferably, the probe is a probe modified at the 5' end with a group which is bound to the solid support of the gene chip, preferably a probe modified with an amino group, more preferably a probe modified with an amino group and a plurality of bases T, and still more preferably a probe modified with an amino group and 16 bases T. After amino modification, the probe can be better connected with a solid phase support (such as a glass slide or a silicon chip) of the gene chip modified by aldehyde group.

Preferably, the PCR reaction system further comprises PCR reaction buffer and/or deoxynucleoside triphosphate (dNTP).

In the present invention, the PCR reaction system may further include a strongly tolerant DNA polymerase. The strongly tolerant DNA polymerase, PCR reaction buffer, deoxynucleotide triphosphate 3 may be obtained commercially, for example in the form of a commercially available 2 XPCR Mix. For example, the 2 × PCR Mix is selected from one or more of the following: 2 XDirect PCR Mix, 2 XTransDirect^TMPCR Super Mix, Phusion Blood Direct PCR Master Mix (2X). Therefore, when the detected sample is whole blood and the like, the whole blood sample does not need any pretreatment, the PCR reaction system in the kit can be directly used for carrying out PCR amplification on the whole blood, and the extraction of genome DNA is not needed, so that the additional purchase of the genome DNA extraction kit is not needed, the cost is saved, the pollution opportunity is reduced, and the kit is suitable for detection of large-scale samples. Thus, in one aspect, the invention also provides a method for directly performing PCR amplification on a sample without pretreatment, but without performing any pretreatmentAnd then hybridizing with the chip to detect the G6PD genotype, a kit, a primer pair, a probe and application thereof.

Reagent	Purchasable factory
		2×Direct PCR Mix	Biometrics bioengineering (Shanghai) Ltd
2×TransDirect^TMPCR SuperMix	Beijing Quanjin Biotechnology Ltd
		Phusion Blood Direct PCR Master Mix(2×)	Thermo Scientific

In a preferred embodiment of the present invention, the amount of the PCR reaction system is as follows:

preferably, the sample to be detected for the kit is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

When the sample to be tested is oral mucosa exfoliated cells, the oral mucosa exfoliated cells are generally suspended in a solvent to be applied to PCR reaction.

Preferably, the kit also comprises a negative probe of the locus rs12979860 of the IL28B gene, and the nucleotide sequence of the negative probe is preferably shown as SEQ ID No.13 (5'-CGAAGGCCAGAACCAGG-3');

preferably, the kit also comprises a negative probe of the rs8099917 site of the IL28B gene, and the nucleotide sequence of the negative probe is preferably shown as SEQ ID No.14 (5'-TCTGTGAGCAAACTCACCCAAAT-3').

Preferably, the kit also comprises a negative probe of the locus rs12980275 of the IL28B gene, and the nucleotide sequence of the negative probe is preferably shown as SEQ ID No.15 (5'-TTCCTAGAAACTCACGTGTCTAA-3').

In a preferred embodiment of the present invention, the final concentrations of the probes having the sequences shown in SEQ ID Nos. 7, 8, 9, 10, 11, 12, 13, 14 and 15 in the PCR reaction system of the kit are 1.43. mu.M, 3.33. mu.M, 1.67. mu.M, 2. mu.M, 2.5. mu.M and 2.5. mu.M, respectively.

Preferably, the kit further comprises a hybridization solution, wherein the hybridization solution preferably comprises a hybridization buffer solution, a pre-hybridization solution, a hybridization reaction solution and a washing solution, the hybridization solution in the hybridization color development kit is preferably selected, and the hybridization color development kit (with the product number of BST03021) produced by Baiao science and technology, Inc. of Shanghai is more preferably selected.

Preferably, the kit further comprises a solution capable of reacting with the detection marker of the primer pair and a color-developing solution such as tetrazolium blue (NBT), 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine salt (BCIP), or Tetramethylbenzidine (TMB). After the PCR product is hybridized with the gene chip and is mixed with a solution which can react with the biological detection markers of the primer pair, the color development solution can react with the solution to develop color so as to detect a hybridization signal.

In the present invention, the PCR may be a PCR technique which is conventional in the art, and may be, for example, a non-fluorescent quantitative PCR.

In order to solve the above technical problems, the present invention aims to provide a primer pair and a probe for detecting IL28B gene, wherein the primer pair and the probe are selected from one or more of the following (1) to (3):

(1) the primer pair and the probe are used for detecting an rs12979860 locus of an IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the probe is shown as SEQ ID NO.7 and/or SEQ ID NO. 8;

(2) the primer pair and the probe are used for detecting a specific amplification primer pair of rs8099917 locus of a gene of IL28B, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.3 and SEQ ID NO.4, and the nucleotide sequence of the probe is shown as SEQ ID NO.9 and/or SEQ ID NO. 10;

(3) the primer pair and the probe are used for detecting a specific amplification primer pair of rs12980275 site of IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.5 and SEQ ID NO.6, and the nucleotide sequence of the probe is shown as SEQ ID NO.11 and/or SEQ ID NO. 12.

Preferably, the probe is in the form of a gene chip, and the probe is immobilized on a solid support of the gene chip.

Preferably, the assay is a PCR assay.

Preferably, the sample to be detected is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

In order to solve the technical problems, the invention aims to provide application of the primer pair and/or the probe in detecting IL28B gene, preparing a reagent or a kit for detecting IL28B gene.

In order to solve the above technical problems, the present invention aims to provide a method for detecting IL28B gene for non-diagnostic purposes, which comprises the following steps: and carrying out PCR reaction on a sample to be detected by using the PCR reaction system, hybridizing the obtained product with the gene chip, and detecting a hybridization signal.

In the invention, the oligonucleotide probe can specifically hybridize with different genotypes of IL28B genes at sites rs12979860, rs8099917 and rs12980275 by a chip hybridization method. The specific primer pair can amplify a target region containing a detection target site. The probe is constructed by the principle that the target DNA and the complementary base of the probe are restored to the original double helix structure by hydrogen bonding.

Preferably, the resulting product is mixed with a hybridization buffer prior to hybridization with the gene chip.

Preferably, the gene chip is prehybridized with a prehybridization solution before hybridizing with the gene chip. This step is advantageous for optimizing the background of chip hybridization.

In the present invention, the detection of the hybridization signal may be to obtain the information to be detected based on the position, intensity, etc. of the labeled signal (the signal of the biological detection marker on the fragment obtained by PCR amplification) on the gene chip, and the method for detecting the hybridization signal may be based on the basic phosphatase-catalyzed tetrazolium blue (NBT) color reaction, horseradish peroxidase-catalyzed 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine salt (BCIP) color reaction, horseradish peroxidase-catalyzed Tetramethylbenzidine (TMB) color reaction and/or fluorescence detection, which is compounded with an avidin antibody, avidin, streptavidin, an anti-digoxigenin antibody, or an anti-fluorescein antibody. The specific method can refer to the gene diagnosis technology compiled by Wangshen five, which comprises the following steps: non-radioactive operating manual. If the amplification product is labeled with a fluorophore, the information to be detected can also be obtained by using a fluorescence detection device (such as a confocal laser scanner Scan array 3000). Preferably, the hybridization signal is detected by alkaline phosphatase-catalyzed tetrazolium blue color reaction, horseradish peroxidase-catalyzed 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine color reaction, horseradish peroxidase-catalyzed tetramethylbenzidine reaction or fluorescence detection.

In the present invention, the gene chip can be prepared according to a conventional method for manufacturing a biochip. For example, if a modified glass slide or silicon wafer is used as the solid support and the 5' -end of the probe contains a poly-dT chain modified with an amino group, the oligonucleotide probe can be prepared as a solution, and then spotted on the modified glass slide or silicon wafer using a spotting device to arrange it into a predetermined sequence or array, followed by standing overnight for immobilization, to obtain a gene chip. If the oligonucleotide probe does not contain amino group modifications, the method of preparation can also be referred to: the "Gene diagnostic technique-non-Radioactive operation Manual" edited by Wangshen five; derisi, JL, equals 1997 in science 278(5338):680 ℃ 686 (Dersi, JL, Iyer VR, Brown PO. expanding the metabolic and genetic control of genomic expression of genes) and the biochip compiled by Malikan et al, chemical industry Press.

In the present invention, the solid phase hybridization between the amplification product and the gene chip is carried out according to the classical method in the art, or according to the "Gene diagnostic technique-nonradioactive operation Manual" compiled by Wangshen five; J. the scientific Press was compiled in molecular cloning guidelines, by Summebruke et al.

The term "strong tolerance" in the case of a strongly tolerant DNA polymerase means that the polymerase is resistant to DNA polymerase inhibitory substances contained in a sample to be tested and is not inhibited by the DNA polymerase inhibitory substances.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

(1) the method, the kit, the primer pair and the probe can be used for detecting the IL28B genotype, have high accuracy, can accurately distinguish various types of mutation sites, and can be used for guiding patients to take medicines.

(2) The gene chip in the kit has good signal-to-noise ratio, is not easily interfered by other interferents in samples to be detected, such as whole blood, oral exfoliated cells and the like, solves the problem that amplification products of the samples to be detected, such as the whole blood, the oral exfoliated cells and the like, containing various interferents influence the hybridization of the chip, and the probe and the primer pair have good specificity.

(3) The method and the kit have the advantages of simple operation and low cost, have high sensitivity (in a preferred embodiment of the invention, the detection limit can be as low as 900 white blood cells), greatly shorten the detection time, have easily-readable and accurate results, are very visual in judgment, and are more suitable for clinical detection and popularization. The method of the invention can preferably take the sample to be detected as the PCR template directly, and can avoid extracting the genome DNA, so that the method does not need to purchase a genome DNA extraction kit additionally, saves the cost, reduces the pollution chance, and is suitable for the detection of large-scale samples. And the required samples are few, only 1 mu L is needed at least, and the samples to be detected such as whole blood and the like are stored in a refrigerator at 4 ℃ for 7 days, so that the detection result is stable, and the reexamination is facilitated, or the samples to be detected such as other detected whole blood and the like of a patient can be utilized, the special sampling of the patient is not needed, and the pain of the patient is relieved. The invention can realize the detection of the IL28B gene by directly carrying out PCR hybridization on a sample to be detected.

(4) The hybridization step in the method can be realized through a full-automatic hybridization process, so that the method is more convenient and quicker and avoids a plurality of uncertain factors existing in the manual operation process.

The method, the kit, the primer pair, the probe and the application thereof detect the IL28B genotype of a patient, and the IL28B gene of the patient is detected, so that the curative effect and the prognosis of the chronic hepatitis C patient on the Peg-IFN/RBV standard treatment scheme are actively predicted, a clinician is guided to select and determine the drug variety and scheme at first, and a scientific basis is provided for individualized medication. In addition, the IL28B genotype can be used as the basis for adjusting the Peg-IFN/RBV standard treatment individualized scheme of the chronic hepatitis C patient, the reason for the failure of the treatment of the scheme is searched, and the method is used for adjusting the variety or the scheme of the medicine, thereby effectively improving the treatment effect of the disease and providing a simple and easy solution.

Drawings

FIG. 1 is a schematic diagram of an IL28B gene chip, in which 0 is a positive quality control probe; 1 is rs12979860C probe; 2 is rs12979860T probe; 12 is an rs12979860 negative quality control probe; 3 is rs8099917T probe; 4 is rs8099917G probe; 34 is rs8099917 negative quality control probe; 5 is rs12980275A probe; 6 is rs12980275G probe; 56 is an rs12980275 negative quality control probe; 00 is blank control.

Fig. 2 is a graph showing the scanning results in example 4.

FIG. 3 is a graph of the results of the sequencing verification in example 4, with the sequencing at the site rs12979860 being shown as an arrow.

FIG. 4 is a graph of the results of sequencing verification in example 4, with the arrow indicating the sequencing of the rs8099917 site.

FIG. 5 is a graph of the results of the sequencing verification in example 4, with the arrow indicating the sequencing of site rs 12980275.

FIG. 6 is a graph showing the results of sensitivity detection in example 5.

FIG. 7 is a graph showing the results of detection in example 6.

FIG. 8 is a graph showing the results of detection in example 7.

FIG. 9 is a graph showing the results of detection in example 8.

Fig. 10 is a graph showing the results of the measurement in the comparative example.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The gene sequence used was derived from NCBI (national center for Biotechnology information).

EXAMPLE 1 preparation of Gene chip

Aldehyde-modified glass slides (product number: BSM03011, Baiao science Co., Ltd., Shanghai). A probe (Biotechnology, Shanghai, Ltd.) was artificially synthesized by dissolving the probe in water to a concentration of 100 pmol/. mu.l, and mixing the solution at an equal ratio (volume ratio) of 2 Xsample application buffer (product No.: BST02010, Shanghai Baiao science and technology, Ltd.). Next, the array of FIG. 1 was spotted using a BD-1 spotter from Baiao science and technology, Shanghai, as described in the specification (each number corresponds to a probe in the bottom of FIG. 1). The mixture was left at room temperature overnight.

The specific probe sequences of each detection site are as follows:

the sequence of the specific oligonucleotide probe for detection site rs12979860C is shown in SEQ ID No.7 (underlined):

NH2-TTTTTTTTTTTTTTTTGAAGGCGCGAACCAGG；

the sequence of the specific oligonucleotide probe for the detection site rs12979860T is shown in SEQ ID No.8 (underlined):

NH2-TTTTTTTTTTTTTTTTCGAAGGCGTGAACCAGG；

the 5 'end of the probe also contains a piece of 16 poly dT (poly deoxythymidylate) modified by 5' amino (-NH 2).

The sequence of the rs12979860 negative control probe of the IL28B gene is shown as SEQ ID No.13 (underlined part):

NH2-TTTTTTTTTTTTTTTTCGAAGGCCAGAACCAGG。

the sequence of the specific oligonucleotide probe of detection site rs8099917T is shown in SEQ ID No.9 (underlined part):

NH2-TTTTTTTTTTTTTTTTTTCTGTGAGCAATTTCACCCAAATT；

the sequence of the specific oligonucleotide probe of detection site rs8099917G is shown in SEQ ID No.10 (underlined):

NH2-TTTTTTTTTTTTTTTTTCTGTGAGCAATGTCACCCAAAT；

The sequence of rs8099917 negative control probe of IL28B gene is shown in SEQ ID NO.14 (underlined part):

NH2-TTTTTTTTTTTTTTTTTCTGTGAGCAAACTCACCCAAAT。

the sequence of the specific oligonucleotide probe for detection site rs12980275A is shown in SEQ ID NO.11 (underlined):

NH2-TTTTTTTTTTTTTTTTAATTCCTAGAAACAGACGTGTCTAA；

the sequence of the specific oligonucleotide probe for detection site rs12980275G is shown in SEQ ID NO.12 (underlined portion):

NH2-TTTTTTTTTTTTTTTTTTCCTAGAAACGGACGTGTCTA；

The sequence of the rs12980275 negative control probe of the IL28B gene is shown in SEQ ID NO.15 (underlined part):

NH2-TTTTTTTTTTTTTTTTTTCCTAGAAACTCACGTGTCTAA。

as shown in FIG. 1, positive quality control probes (purchased from Baiao science and technology Co., Ltd., Shanghai), rs12979860C probe (SEQ ID NO.7), rs12979 12979860T probe (SEQ ID NO.8), rs8099917T probe (SEQ ID NO.9), rs8099917G probe (SEQ ID NO.10), rs12980275A probe (SEQ ID NO.11), rs12980275G probe (SEQ ID NO.12), rs12979860 negative control probe (SEQ ID NO.13) of IL28B gene, rs8099917 negative control probe (SEQ ID NO.14) of IL28B gene, rs12980275 negative control probe (SEQ ID NO.15) of IL28B gene, and blank control were spotted on a glass slide; and (5) preparing the gene chip. Wherein 0 is a positive quality control probe; 1 is rs12979860C probe; 2 is rs12979860T probe; 12 is an rs12979860 negative quality control probe; 3 is rs8099917T probe; 4 is rs8099917G probe; 34 is rs8099917 negative quality control probe; 5 is rs12980275A probe; 6 is rs12980275G probe; 56 is an rs12980275 negative quality control probe; 00 is blank control.

Example 2 amplification of IL28B Gene fragment by PCR Using the primers provided by the invention

Primers were synthesized by Compton Biotechnology engineering (Shanghai) Ltd, and the primer information was as follows.

rs12979860 upstream primer sequence SEQ ID No. 1:

5’-AGTCTGGGATTCCTGGACGTGG-3’，

rs12979860 downstream primer sequence SEQ ID NO. 2:

5’-GGCTCAGGGTCAATCACAGAAGGGAG-3’，

rs8099917 upstream primer sequence SEQ ID No. 3:

5’-CCCACTTCTGGAACAAATCG-3’，

rs8099917 downstream primer sequence SEQ ID No. 4:

5’-TGGTATCAACCCCACCTCAA-3’，

rs12980275 upstream primer sequence SEQ ID No. 5:

5’-AGAGAGCAAGAGGAGGGAAG-3’，

rs12980275 downstream primer sequence SEQ ID No. 6:

5’-CTGGTCCTAGTGGTGTTTGC-3’，

and, the 5' end of the primer is modified with biotin.

Then dissolved in water and diluted to a concentration of 10 pmol/. mu.l.

The PCR amplification system was formulated as shown in Table 1 below:

TABLE 1

Reagent	Addition amount (μ l)
		Blood template (Whole blood, without any pretreatment)	1
rs12979860 upstream primer (10. mu.M)	0.35～1.75
		rs12979860 downstream primer (10. mu.M)	1.75
rs8099917 upstream primer (10. mu.M)	0.5～1.5
		rs8099917 downstream primer (10. mu.M)	1.5
rs12980275 upstream primer (10. mu.M)	0.55～2.1
		rs12980275 downstream primer (10. mu.M)	2.1
2×TransDirect^TM PCR SuperMix	12.5
		ddH₂O	To 25

Amplification was performed using a PCR apparatus (TC-96/G/H (b) PCR amplification apparatus, available from Bori science, Inc., Hangzhou) according to the following procedure:

50℃5min；95℃10min；{95℃20sec，64℃(-1℃/cycle)30sec，72℃30sec，10cycles}；{95℃20sec，54℃30sec，72℃30sec，35cycles}；72℃5min。

the SNP sequence of the rs12979860 locus of the IL28B gene is derived from the SNP database information of NCBI and is a partial sequence of IL28B, and a target product amplified by the invention is contained in the sequence.

The SNP sequence of rs8099917 site of IL28B gene is obtained from NCBI SNP database information and is a partial sequence of IL28B, and a target product amplified by the invention is contained in the sequence.

The SNP sequence of the rs12980275 site of the IL28B gene is obtained from the SNP database information of NCBI, is a partial sequence of IL28B, and a target product amplified by the invention is contained in the sequence.

Example 3 hybridization of the Gene chip obtained in example 1 and the PCR product obtained in example 2

Hybridization was performed in a fully automatic hybridization apparatus (BSE 03011) using the hybridization color development kit (BST03021) available from shanghai baiao science and technology gmbh, as follows: the hybridization color development kit is alkaline phosphatase color development reaction.

1) Preparing a hybridization reaction solution: 185. mu.L of the hybridization buffer in the hybridization color development kit was aspirated, 15. mu.L of the amplification product of example 2 was added thereto, and the mixture was mixed well.

2) The gene chip prepared in example 1 and the PCR product prepared in example 2 were placed on a hybridization apparatus and covered tightly.

3) Setting a hybridization program: prehybridization solution, 44 ℃, 5 min; hybridizing the reaction solution at 44 ℃ for 30 min; washing at 1, 44 deg.C for 6 min; washing at 1, 44 deg.C for 6 min; washing at 2, 28 deg.C for 5 min; washing at 2, 28 deg.C for 5 min; antibody solution, 28 deg.C, 20 min; washing at 2, 28 deg.C for 5 min; washing at 2, 28 deg.C for 5 min; washing at 28 deg.C for 3 min; developing solution at 44 ℃ for 30 min; pre-hybridization solution at 28 ℃ for 2 min; pre-hybridization solution at 28 ℃ for 2 min; the specific operation is shown in the instruction book of the instrument. The program is operated, and the hybridization color reaction is automatically carried out.

Example 4 detection results of hybridization signals of Gene chip

The hybridized and washed gene chip was placed on a BaiO BE3.0 biochip reader (Shanghai Baiao science and technology Co., Ltd.) and scanned to obtain the detection result shown in FIG. 2, which was very visual. The result shows that the IL28B genotype of the subject belongs to rs12979860CC/rs8099917TT/rs12980275AA genotype, the detection result is verified by sequencing, and the sequencing results are shown in fig. 3, fig. 4 and fig. 5, and the results completely conform to each other.

Example 5 results of sensitivity detection

1) Sample preparation

Taking IL28B genes rs12979860CC, rs8099917TT and rs12980275AA type blood (IL-00 for short), IL28B genes rs12979860CT, rs8099917TG and rs12980275AG type blood (IL-01 for short), providing blood samples with known genotypes by clinical cooperative units, diluting various blood samples by using negative control, and diluting to obtain samples with different leukocyte contents as shown in the following table 2:

TABLE 2

Name (R)	Leukocyte concentration(10⁹/L)	Name (R)	Leukocyte concentration (10)⁹/L)
				IL-00-1	2	IL-01-1	2
IL-00-2	1	IL-01-2	1
				IL-00-3	0.9	IL-01-3	0.9

2) Detection of

The samples were tested as described in examples 1-4.

3) Results

The hybridization results are shown in FIG. 6: wherein FIG. A, B, C, D, E, F corresponds to IL-00-1, IL-00-2, IL-00-3, IL-01-1, IL-01-2, and IL-01-3, respectively.

The detection method has high detection sensitivity and can detect 0.9 multiplied by 10⁹Blood at a leukocyte concentration of one/L, i.e. a sensitivity of 900 leukocytes.

Example 6 interference test results

1) Sample preparation

Selecting EDTA anticoagulant whole blood samples (provided by clinical cooperative unit and all known genotype blood samples) known as IL28B gene rs12979860CC, rs8099917TT and rs12980275AA type blood (IL-00) and preparing a sample containing the interferent according to the following method: according to the solubility of the interferent in the blood matrix, the easily soluble substance is prepared by directly adding the interferent solid into the blood, dissolving and uniformly mixing; for insoluble matters, dissolving the insoluble matters by adopting a proper solvent, adding the dissolved matters into a matrix, and uniformly mixing the dissolved matters; samples with different levels of interferents are shown in table 3 below:

TABLE 3

Name of interfering substance	Concentration of
		Total Cholesterol	250mg/dL
Triglyceride	3000mg/dL
		Bilirubin	20mg/dL

2) Detection of

The above samples were tested by the gene chip according to the methods of examples 1 to 4.

3) Results

The hybridization results are shown in FIG. 7: wherein, FIG. G, H, I corresponds to total cholesterol, triglyceride, and bilirubin, respectively.

250mg/dL total cholesterol in blood; 3000mg/dL triglyceride; 20mg/dL bilirubin does not interfere with the assay of the present invention.

Example 7 detection results under different primer concentration conditions

The IL28B genes rs12979860CC, rs8099917TT and rs12980275AA type blood and amplification solutions with different primer concentration ratios are taken as shown in the following table 4, and detection is carried out according to the methods of the embodiments 1-4. The detection results are shown in fig. 8.

TABLE 4

	Primer mix 1	Primer ratio 2	Primer ratio 3
				rs12979860 upstream primer (μ M)	0.14	0.16	0.12
rs12979860 downstream primer (μ M)	0.70	0.72	0.60
				rs8099917 upstream primer (μ M)	0.20	0.20	0.20
rs8099917 downstream primer (μ M)	0.60	0.64	0.60
				rs12980275 upstream primer (μ M)	0.22	0.20	0.20
rs12980275 downstream primer (μ M)	0.84	0.76	0.72

From the above results, the concentrations of the upstream primer and the downstream primer can be determined, the primer concentrations can be in the range of 0.12-0.84. mu.M, preferably the rs12979860 upstream primer and the downstream primer are 0.14. mu.M and 0.70. mu.M, preferably the rs8099917 upstream primer and the downstream primer are 0.20. mu.M and 0.60. mu.M, preferably the rs12980275 upstream primer and the downstream primer are 0.22. mu.M and 0.84. mu.M.

Example 8 test results at different Probe concentrations

The gene chips of IL28B gene rs12979860CC, rs8099917TT and rs12980275AA type blood and different probe concentration ratios are taken as shown in the following table 5, and detection is carried out according to the methods of the embodiments 1-4. The detection results are shown in fig. 9.

TABLE 5

	Probe ratio 1	Probe ratio 2
			SEQ ID NO.7(μM)	1.43	2.00
SEQ ID NO.8(μM)	3.33	5.00
			SEQ ID NO.9(μM)	1.67	2.50
SEQ ID NO.10(μM)	1.67	2.50
			SEQ ID NO.11(μM)	2.00	3.33
SEQ ID NO.12(μM)	2.50	3.33

The probe of SEQ ID NO.7 is rs12979860C, the probe of SEQ ID NO.8 is rs12979860T, the probe of SEQ ID NO.9 is rs8099917T, the probe of SEQ ID NO.10 is rs8099917G, the probe of SEQ ID NO.11 is rs12980275A, and the probe of SEQ ID NO.12 is rs 12980275G.

Combining the above results, the concentrations of the rs12979860C probe, the rs12979860T probe, the rs8099917T probe, the rs8099917G probe, the rs12980275A probe and the rs12980275G probe are preferably 1.43. mu.M, 3.33. mu.M, 1.67. mu.M, 2.0. mu.M and 2.5. mu.M.

Comparative example

1) Control primer pair 1 (for locus rs 12979860):

the upstream primer SEQ ID NO. 16: 5'-TGTACTGAAC CAGGGAG-3'

The downstream primer is SEQ ID NO. 17: 5'-GTTCCCATACACCCGTTCCT-3'

The product is as follows: 260bp

Comparison primer pair 2 (for rs12979860 locus)

The upstream primer SEQ ID NO. 18: 5'-CGCTTATCGCATACGGCTAGGC-3'

The downstream primer is SEQ ID NO. 19: 5'-GGAGCGCGGAGTGCAATT-3'

The product is as follows: 167bp

2) And (3) probe:

detection of rs12979860C design failure probe SEQ ID NO.20 (underlined):

NH2-TTTTTTTTTTTTTTTTGAAGGCGCGAACCA

detection of the rs12979860T design failure probe SEQ ID NO.21 (underlined):

NH2-TTTTTTTTTTTTTTTTCGAAGGCGTGAACCA

detection of rs8099917T design failure probe SEQ ID No.22 (underlined):

NH2-TTTTTTTTTTTTTTTTTGAGCAATTTCACCCAAATT

detection of rs8099917G design failure probe SEQ ID No.23 (underlined):

NH2-TTTTTTTTTTTTTTTTTGAGCAATGTCACCCAAAT

detection of rs12980275A design failure probe SEQ ID NO.24 (underlined):

NH2-TTTTTTTTTTTTTTTTCCTAGAAACAGACGTGTCTAA

detection of rs12980275G design failure probe SEQ ID NO.25 (underlined):

NH2-TTTTTTTTTTTTTTTTTAGAAACGGACGTGTCTA

the amplification solution and the chip are prepared by using the above-mentioned comparison primer pair 1 and comparison primer pair 2 and the above-mentioned probe respectively, and the whole blood (provided by clinical cooperative units and all known genotype blood samples) of the genotypes rs12979860CC, rs8099917TT and rs12980275AA of the IL28B gene is used as a sample, and the test is carried out according to the examples 1-4, and the detection pictures are respectively shown as the left picture and the right picture of fig. 10 (the left picture and the right picture are the detection results of the same sample, wherein the comparison primer pair 1 is used for the rs12979860 locus in the left picture, the probe is used for the above-mentioned probe SEQ ID NO.20-SEQ ID NO.25, and the comparison primer pair 2 is used for the rs12979860 locus in the right picture, and the probe is used for the above-mentioned probe SEQ ID NO.20-SEQ ID NO. 25). As can be seen from the figure, the signals of the gene chip were weak and the genotype could not be normally judged in both cases of the combination of the control primer set 1 and the control probe and the combination of the control primer set 2 and the control probe.

SEQUENCE LISTING

<110> Shanghai Baiao science and technology, Inc

<120> method, kit, primer set and probe for detecting IL28B gene

<130>P19014698C

<160>25

<170>PatentIn version 3.5

<210>1

<211>22

<212>DNA

<213>Artificial Sequence

<220>

<223> rs12979860 upstream primer sequence

<400>1

agtctgggat tcctggacgt gg 22

<210>2

<211>26

<212>DNA

<213>Artificial Sequence

<220>

<223> rs12979860 downstream primer sequence

<400>2

ggctcagggt caatcacaga agggag 26

<210>3

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> rs8099917 upstream primer sequence

<400>3

cccacttctg gaacaaatcg 20

<210>4

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> rs8099917 downstream primer sequence

<400>4

tggtatcaac cccacctcaa 20

<210>5

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> rs12980275 upstream primer sequence

<400>5

agagagcaag aggagggaag 20

<210>6

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> rs12980275 downstream primer sequence

<400>6

ctggtcctag tggtgtttgc 20

<210>7

<211>16

<212>DNA

<213>Artificial Sequence

<220>

<223> specific oligonucleotide probe for detection site rs12979860C

<400>7

gaaggcgcga accagg 16

<210>8

<211>17

<212>DNA

<213>Artificial Sequence

<220>

<223> specific oligonucleotide probe for detection site rs12979860T

<400>8

cgaaggcgtg aaccagg 17

<210>9

<211>25

<212>DNA

<213>Artificial Sequence

<220>

<223> specific oligonucleotide probe for detection site rs8099917T

<400>9

ttctgtgagc aatttcaccc aaatt 25

<210>10

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<223> specific oligonucleotide probe for detection site rs8099917G

<400>10

tctgtgagca atgtcaccca aat 23

<210>11

<211>25

<212>DNA

<213>Artificial Sequence

<220>

<223> specific oligonucleotide probe for detection site rs12980275A

<400>11

aattcctaga aacagacgtg tctaa 25

<210>12

<211>22

<212>DNA

<213>Artificial Sequence

<220>

<223> specific oligonucleotide probe for detection site rs12980275G

<400>12

ttcctagaaa cggacgtgtc ta 22

<210>13

<211>17

<212>DNA

<213>Artificial Sequence

<220>

<223> rs12979860 negative control probe of IL28B gene

<400>13

cgaaggccag aaccagg 17

<210>14

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<223> rs8099917 negative control probe of IL28B gene

<400>14

tctgtgagca aactcaccca aat 23

<210>15

<211>23

<212>DNA

<213>Artificial Sequence

<220>

<223> IL28B gene rs12980275 bit negative control probe

<400>15

ttcctagaaa ctcacgtgtc taa 23

<210>16

<211>17

<212>DNA

<213>Artificial Sequence

<220>

<223> control primer set 1 (for rs12979860 site) upstream primer

<400>16

tgtactgaac cagggag 17

<210>17

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> comparison primer set 1 (for rs12979860 site) downstream primer

<400>17

gttcccatac acccgttcct 20

<210>18

<211>22

<212>DNA

<213>Artificial Sequence

<220>

<223> control primer set 2 (for rs12979860 site) upstream primer

<400>18

cgcttatcgc atacggctag gc 22

<210>19

<211>18

<212>DNA

<213>Artificial Sequence

<220>

<223> control primer set 2 (for rs12979860 site) downstream primer

<400>19

ggagcgcgga gtgcaatt 18

<210>20

<211>14

<212>DNA

<213>Artificial Sequence

<220>

<223> detection of rs12979860C design failure probes

<400>20

gaaggcgcga acca 14

<210>21

<211>15

<212>DNA

<213>Artificial Sequence

<220>

<223> detection of rs12979860T design failure probes

<400>21

cgaaggcgtg aacca 15

<210>22

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> detection of rs8099917T design failure probe

<400>22

tgagcaattt cacccaaatt 20

<210>23

<211>19

<212>DNA

<213>Artificial Sequence

<220>

<223> detection of rs8099917G design failure probe

<400>23

tgagcaatgt cacccaaat 19

<210>24

<211>21

<212>DNA

<213>Artificial Sequence

<220>

<223> detection rs12980275A design failure probe

<400>24

cctagaaaca gacgtgtcta a 21

<210>25

<211>18

<212>DNA

<213>Artificial Sequence

<220>

<223> detection rs12980275G design failure probe

<400>25

tagaaacgga cgtgtcta 18

Claims

1. A kit for detecting IL28B gene, which is characterized in that the kit comprises a PCR reaction system and a gene chip, wherein the PCR reaction system comprises a primer pair consisting of an upstream primer and a downstream primer, and the gene chip comprises a probe; wherein the primer pair and the probe are selected from any one, two or three of the following (1) to (3):

(1) the primer pair and the probe are used for detecting an rs12979860 locus of an IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and/or the nucleotide sequence of the probe is shown as SEQ ID NO.7 and/or SEQ ID NO. 8;

(2) the primer pair and the probe are used for detecting rs8099917 site of IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.3 and SEQ ID NO.4, and/or the nucleotide sequence of the probe is shown as SEQ ID NO.9 and/or SEQ ID NO. 10;

(3) the primer pair and the probe are used for detecting the rs12980275 site of the IL28B gene, wherein the nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID NO.5 and SEQ ID NO.6, and/or the nucleotide sequence of the probe is shown as SEQ ID NO.11 and/or SEQ ID NO. 12.

2. The kit according to claim 1, wherein the concentration of the primer pair in the PCR reaction system is 0.12 to 0.84. mu.M; preferably:

in the step (1), the concentration of the primer pair is 0.12-0.72 mu M, and the concentration of the upstream primer is preferably 0.14 mu M; and/or, the concentration of the downstream primer is preferably 0.6 μ M;

and/or, in the step (2), the concentration of the primer pair is 0.2-0.64 mu M, and the concentration of the upstream primer is preferably 0.2 mu M; and/or, the concentration of the downstream primer is preferably 0.6 μ M;

and/or, in the step (3), the concentration of the primer pair is 0.2-0.84 mu M, and the concentration of the upstream primer is preferably 0.22 mu M; and/or, the concentration of the downstream primer is preferably 0.84. mu.M.

3. The kit of claim 1 or 2, wherein the concentration of the probes in the gene chip is 1.25 to 5 μ M;

preferably, the concentration of the probe for detecting the rs12979860 locus of the IL28B gene is 1.4-5 mu M, and preferably 3 mu M; and/or the concentration of the probe for detecting the rs8099917 site of the IL28B gene is 1.25-2.5 mu M, preferably 1.67 mu M; and/or the concentration of the probe for detecting the locus rs12980275 of the IL28B gene is 1.43-3.3 mu M, preferably 2.5 mu M.

4. The kit according to any one of claims 1 to 3, wherein the primer pair is modified at the 5' end by a biological detection marker, and the biological detection marker is preferably biotin, digoxin, fluorescein, a fluorescein derivative, a fluorescent molecule, alkaline phosphatase or horseradish peroxidase;

and/or, the probe is a probe whose 5' end is modified by a group, the group is combined with a solid phase support of the gene chip, preferably a probe modified by an amino group, more preferably a probe modified by an amino group and a plurality of bases T, and still more preferably a probe modified by an amino group and 16 bases T; the modifier of the solid phase support is preferably aldehyde group;

and/or, the PCR reaction system also comprises a strong tolerance DNA polymerase, and the strong tolerance DNA polymerase is preferably Direct PCR Mix and Trans Direct^TMOne or more of PCR Super Mix, Phusion Blood Direct PCR Mastermix;

and/or the sample to be detected for the kit is a biological sample containing genomic DNA, preferably whole blood or oral mucosa exfoliative cells.

5. The kit of any one of claims 1 to 4, wherein the PCR reaction system further comprises a PCR reaction buffer and/or deoxynucleoside triphosphates;

and/or the kit further comprises a negative probe of an IL28B gene rs12979860 site and/or a negative probe of an IL28B gene rs8099917 site and/or a negative probe of an IL28B gene rs12980275 site; preferably:

the nucleotide sequence of the negative probe of the site rs12979860 of the IL28B gene is shown as SEQ ID No. 13; and/or the nucleotide sequence of the negative probe of the rs8099917 site of the IL28B gene is shown as SEQ ID No. 14; and/or the nucleotide sequence of the negative probe of the site rs12980275 of the IL28B gene is shown as SEQ ID No. 15.

6. The kit according to any one of claims 1 to 5, wherein the kit further comprises a hybridization solution, preferably comprising a hybridization buffer, a prehybridization solution and/or a wash solution; and/or, the kit further comprises a solution capable of reacting with the detection marker of the primer pair and a color developing solution such as tetrazolium blue, 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine salt, or tetramethylbenzidine.

7. A primer pair and a probe for detecting IL28B gene, wherein the primer pair and the probe are selected from one or more of the following (1) to (3):

8. The primer pair and probe according to claim 7, wherein the probe is included in a gene chip, and the probe is immobilized on a solid support of the gene chip; and/or, the detection is PCR detection; and/or the detected sample is a biological sample containing genome DNA, preferably whole blood or oral mucosa exfoliative cells.

9. Use of the primer pair and/or the probe according to claim 7 or 8 for detecting IL28B gene, for preparing a reagent or a kit for detecting IL28B gene.

10. A method for detecting IL28B gene for non-diagnostic purposes, comprising the steps of: carrying out PCR reaction on a sample to be detected by using the PCR reaction system of any one of claims 1 to 6, hybridizing the obtained product with the gene chip of any one of claims 1 to 6, and detecting a hybridization signal; preferably:

mixing the obtained product with a hybridization buffer solution before hybridization with the gene chip;

and/or, the gene chip is pre-hybridized with a pre-hybridization solution before being hybridized with the gene chip;

and/or the method for detecting the hybridization signal is tetrazolium blue color development reaction catalyzed by alkaline phosphatase, 5-bromo-4-chloro-3-indoxyl-phosphate-4-toluidine color development reaction catalyzed by horseradish peroxidase, tetramethylbenzidine reaction catalyzed by horseradish peroxidase and/or fluorescence detection;

and/or the detected sample is a biological sample containing genome DNA, preferably whole blood or oral mucosa exfoliative cells.