CN107937507B - Soluble microneedle patch and preparation method thereof - Google Patents
Soluble microneedle patch and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a soluble microneedle patch and a preparation method thereof. The invention firstly provides a complete set of probes, which consist of single-stranded DNA molecules shown in a sequence 1-a sequence 9 of a sequence table. The invention also protects the gene chip obtained by fixing the probe set on the chip medium. The gene chip can detect the mutation condition of the HBsAg locus in a sample to be detected. During detection, virus DNA is extracted from a serum sample of a patient to be detected, the virus DNA is hybridized with a chip probe after in vitro amplification, and the mutation type is judged according to a hybridization signal. The detection method provided by the invention has the advantages of high throughput, high specificity, simple operation, automatic result judgment and easy standardized control, is suitable for clinical detection application of HBsAg genetic variation, and can meet the requirements of physical examination and donor screening.
Description
Technical Field
The invention relates to a soluble microneedle patch and a preparation method thereof.
Background
Hepatitis b is caused by infection with Hepatitis B Virus (HBV). HBV belongs to the hepadnaviridae, is a circular double-strand DNA virus, has an outer layer of a lipoprotein envelope and contains hepatitis B surface antigen (HBsAg); the inner part is a core and contains nucleic acid and hepatitis B core antigen.
The hepatitis B virus S gene encodes the surface antigen (HBsAg). HBsAg has important biological significance, is a main component of hepatitis B vaccine, and is an important basis for diagnosing hepatitis B virus infection. The S gene region can be divided into 3 segments of an S region, a previous S1 region and a previous S2 region, wherein 5' ends of the 3 segments respectively have an initiation codon (AUG), but share a stop codon. The translated products form the main protein (226 amino acids), the middle protein (281 amino acids) and the large protein (409 amino acids) of HBsAg. Mutation of the S gene region, particularly mutation of the alpha determinant amino acid 145 site, can cause antigenic change of the HBsAg, so that HBsAg diagnostic reagents widely used in clinic cannot be detected, detection omission is caused, and virus carriers can become blood donors and bring serious consequences to blood recipients.
The current molecular detection methods for HBsAg gene variation mainly comprise 4 types: (1) sequencing method: the gold standard of HBV gene detection has high accuracy, but lower sensitivity and long time consumption; (2) the RFLP method comprises the following steps: the principle is that a DNA fragment to be detected is cut by restriction enzyme to identify and cut a specific sequence, the product after the enzyme cutting is subjected to electrophoresis, whether a target gene has the specific sequence or not is judged according to the size of the DNA fragment, but the standardization degree is low, the detection capability on mixed genotypes is poor, and when the gene mutation is detected, a plurality of detection systems are required to respectively detect the mutation with more than 1 base; (3) the quantitative PCR method comprises the following steps: the sensitivity is high, but the flux is low, and the multi-gene locus variation is difficult to detect; (4) reverse linear probe hybridization method: firstly, known probes are respectively spotted on a nitrocellulose membrane or a nylon membrane, hybridized with a PCR amplification product of a biotin primer marked at the 5' end, and then a hybridization signal is displayed through corresponding color reaction. The judgment by naked eyes is easy to misjudge, the detection cost is high, and the routine development in the domestic clinical laboratory is difficult.
Disclosure of Invention
The invention aims to provide a soluble microneedle patch and a preparation method thereof.
The invention firstly provides a probe set, which consists of a probe P1, a probe P2, a probe P3, a probe P4, a probe P5, a probe P6, a probe P7, a probe P8 and a probe P9;
the probe P1 is (a1) or (a2) as follows:
(a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;
(a2) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and have the same functions as the sequence 1;
the probe P2 is (a3) or (a4) as follows:
(a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;
(a4) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and have the same functions as the sequence 2;
the probe P3 is (a5) or (a6) as follows:
(a5) a single-stranded DNA molecule shown in sequence 3 of the sequence table;
(a6) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and have the same functions as the sequence 3;
the probe P4 is (a7) or (a8) as follows:
(a7) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;
(a8) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and having the same functions as the sequence 4;
the probe P5 is (a9) or (a10) as follows:
(a9) a single-stranded DNA molecule shown in sequence 5 of the sequence table;
(a10) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and having the same functions as the sequence 5;
the probe P6 is (a11) or (a12) as follows:
(a11) a single-stranded DNA molecule shown in sequence 6 of the sequence table;
(a12) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same functions as the sequence 6;
the probe P7 is (a13) or (a14) as follows:
(a13) a single-stranded DNA molecule shown in sequence 7 of the sequence table;
(a14) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 7 and having the same functions as the sequence 7;
the probe P8 is (a15) or (a16) as follows:
(a15) a single-stranded DNA molecule shown in sequence 8 of the sequence table;
(a16) DNA molecules which are obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 8 and have the same functions as the sequence 8;
the probe P9 is (a17) or (a18) as follows:
(a17) a single-stranded DNA molecule shown in sequence 9 of the sequence table;
(a18) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 9 and has the same function as the sequence 9.
The probe P1, the probe P2, the probe P3, the probe P4, the probe P5, the probe P6, the probe P7, the probe P8 and the probe P9 are subjected to NH reaction2And (5) modifying. The NH2The modification may be at the 5 'end or the 3' end of the probe. In an embodiment of the invention, the NH2The modification is located at the 3' end of the probe.
The probe set also comprises a quality control probe and a negative probe. The quality control probe can be specifically shown as a sequence 12 in a sequence table. The negative probe can be a plant gene sequence irrelevant to hepatitis B virus, and can be specifically shown as a sequence 13 in a sequence table. The 5 'end of the quality control probe is labeled by biotin, and the 3' end is labeled by NH2And (5) modifying.
The invention also protects the application of the probe set, which is (b1) or (b 2):
(b1) detecting HBsAg site mutation in a sample to be detected;
(b2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
The invention also protects the gene chip, which is obtained by fixing the probe set on a chip medium.
The chip medium can be an aldehyde glass sheet.
The preparation method of the gene chip specifically comprises the following steps: each probe in the set of probes was dissolved in a spotting solution (3 XSSC, 0.01% SDS), spotted on the surface of an aldehyde-based glass plate using a spotting instrument, and dried overnight. The concentration of each probe in the spotting fluid may specifically be 100. mu.M.
The invention also protects the application of the gene chip, which is (c1) or (c 2):
(c1) detecting HBsAg site mutation in a sample to be detected;
(c2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
The invention also protects a primer chip combination, which consists of the gene chip and a primer pair;
the primer pair consists of a primer F and a primer R;
the primer F is (d1) or (d 2):
(d1) a single-stranded DNA molecule shown in sequence 10 of the sequence table;
(d2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 10 and has the same function as the sequence 10;
the primer R is (d3) or (d 4):
(d3) a single-stranded DNA molecule shown in sequence 11 of the sequence table;
(d4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 11 and has the same function as the sequence 11.
And the primer R is labeled by biotin. The biotin label may be located at the 5 'end or the 3' end of primer R. In an embodiment of the invention, the biotin label is located at the 5' end of primer R.
The invention also protects a primer probe combination, which consists of the complete set of probes and a primer pair;
the primer pair consists of a primer F and a primer R;
the primer F is (d1) or (d 2):
(d1) a single-stranded DNA molecule shown in sequence 10 of the sequence table;
(d2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 10 and has the same function as the sequence 10;
the primer R is (d3) or (d 4):
(d3) a single-stranded DNA molecule shown in sequence 11 of the sequence table;
(d4) DNA molecules obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 11 and having the same functions as the sequence 11;
and the primer R is labeled by biotin. The biotin label may be located at the 5 'end or the 3' end of primer R. In an embodiment of the invention, the biotin label is located at the 5' end of primer R.
The invention also protects the application of the primer chip combination or the primer probe combination, which is (e1) or (e 2):
(e1) detecting HBsAg site mutation in a sample to be detected;
(e2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
The invention also protects a kit A containing the set of probes, or a kit B containing the gene chip, or a kit C containing the primer chip combination, or a kit D containing the primer probe combination; the application of the kit A or the kit B or the kit C or the kit D is (f1) or (f 2):
(f1) detecting HBsAg site mutation in a sample to be detected;
(f2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
The invention also provides a method for detecting HBsAg site mutation in a sample to be detected, which comprises the following steps: taking the nucleic acid of the sample to be detected in vitro as a template, and carrying out PCR amplification by adopting the primer pair to obtain a PCR amplification product; and hybridizing the PCR amplification product with the gene chip, and judging the mutation of the HBsAg locus in the sample to be detected according to the hybridization result.
In the method, the template can be prepared according to the following steps: extracting nucleic acid of an isolated sample to be detected, and performing nested PCR amplification by using the nucleic acid as a template to obtain an amplification product (the template). The nested PCR is divided into two rounds of amplification, the first round of amplification takes the nucleic acid as a template, and adopts a primer 1 and a primer 2 to carry out PCR amplification, and the second round of amplification takes a PCR amplification product obtained in the first round as a template, and adopts a primer 3 and a primer 4 to carry out PCR amplification. The primer 1 is a single-stranded DNA molecule shown as a sequence 15 in a sequence table. The primer 2 is a single-stranded DNA molecule shown as a sequence 16 in a sequence table. The primer 3 is a single-stranded DNA molecule shown in a sequence 17 of a sequence table. The primer 4 is a single-stranded DNA molecule shown as a sequence 18 in a sequence table. The reaction procedure of the first round of amplification can be specifically as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 35s, annealing at 59 ℃ for 35s, extension at 72 ℃ for 70s, and 2 cycles; denaturation at 94 ℃ for 35s, annealing at 57 ℃ for 35s, and extension at 72 ℃ for 70s, for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 70s for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 53 ℃ for 35s, and extension at 72 ℃ for 70s for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 51 ℃ for 35s, and extension at 72 ℃ for 70s, for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 70s for 30 cycles; extension at 72 ℃ for 7 min. The reaction procedure of the second round of amplification can be specifically pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 25s, annealing at 59 ℃ for 25s, extension at 72 ℃ for 50s, and 30 cycles; extension at 72 ℃ for 7min and at 68 ℃ for 5 min.
The nucleic acid may specifically be viral DNA.
In the method, a biochip imager can be used to scan the gene chip to obtain the hybridization result.
In the method, before the PCR amplification product is hybridized with the complete set of probes or the gene chip, the PCR amplification product can be thermally denatured at 98 ℃ for 5min, and then placed in an ice bath for 5 min.
The hybridization comprises the following steps: (1) hybridizing the PCR product with the gene chip at 45 ℃ for 1 h; (2) horse radish peroxidase labeled streptavidin was added to the chip and hybridized in a 37 ℃ water bath for 25 min.
The hybridization specifically comprises the following steps: (a) mixing the PCR product with hybridization solution (2 XSSC, 1.2% SDS, 5% formamide) uniformly, adding the mixture into a sample application area of a gene chip, and hybridizing the mixture in a water bath at 45 ℃ for 1 hour; (b) after the step (a) is finished, taking the gene chip, sequentially washing the gene chip by respectively using solution A (1 XSSC, 0.2% SDS), solution B (0.2 XSSC) and solution C (0.1 XSSC) for 20 seconds, and spin-drying; (c) after the step (b) is finished, taking the gene chip, adding a marking solution (horse radish peroxidase marked streptavidin) into the sample application area, and hybridizing in water bath at 37 ℃ for 25 min; (d) after the step (c) is completed, the gene chip is taken out and washed for 20s by 1 XPBST (1 XPBST + 0.05% Tween 20) and dried.
Any one of the HBsAg sites specifically includes the following 4 HBsAg sites: (e1) HBsAg amino acid 118; (e2) HBsAg amino acid 120; (e3) HBsAg amino acid 126; (e4) HBsAg amino acid 145.
Any HBsAg site mutation is specifically the following 4 mutations: (f1) the 118 th amino acid of the HBsAg is mutated from threonine to other amino acid (specifically lysine); (f2) HBsAg120 amino acid is mutated from proline to other amino acid (specifically glutamine); (f3) the 126 th amino acid of the HBsAg is mutated from threonine to other amino acid (specifically isoleucine); (f3) HBsAg amino acid 145 is mutated from glycine to another amino acid (specifically alanine or arginine).
When any one of the complete set of probes or the gene chip or the primer probe combination or the primer chip combination is used for detecting HBsAg site mutation, the result judgment method comprises the following steps: the positive quality control probe and the negative quality control probe indicate that the result is effective, and the effective result reading method comprises the following steps: a positive probe P1 indicates that the HBsAg has wild type (threonine) at amino acid 118; a positive probe P2 indicates that the HBsAg has a mutant form (lysine) at amino acid 118; a positive probe P3 indicates that the HBsAg has wild type amino acid 120 (proline); a positive probe P4 indicates that HBsAg has a mutant type (glutamine) at amino acid 120; a positive probe P5 indicates that the 126 th amino acid of HBsAg is wild type (threonine); a positive probe P6 indicates that the 126 th amino acid of HBsAg is mutant (isoleucine); a positive probe P7 indicates that the HBsAg has wild type (glycine) at amino acid number 145; a positive probe P8 indicates that the HBsAg has a mutant form (alanine) at amino acid position 145; a positive probe for probe P9 indicates that the HBsAg has a mutant form (arginine) at amino acid position 145.
The wild type of any HBsAg is shown as a sequence 14 in a sequence table.
Any one of the above samples to be tested may specifically be serum.
The invention utilizes the high-flux and parallel detection characteristics of the gene chip to design probes for a plurality of genes causing HBsAg mutation, and the probes are combined on the same chip; during detection, virus DNA is extracted from a serum sample of a patient to be detected, the virus DNA is hybridized with a chip probe after in vitro amplification, and the mutation type is judged according to a hybridization signal. The detection method provided by the invention has the advantages of high throughput, high specificity, simple operation, automatic result judgment and easy standardized control, is suitable for clinical detection application of HBsAg genetic variation, and can meet the requirements of physical examination and donor screening.
Drawings
FIG. 1 is a gene chip map.
FIG. 2 is an electrophoretogram of PCR amplification products obtained in step 2 from a part of patient samples in example 3. M: marker, the strip size is from top to bottom in proper order: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp; 1. 2: negative control (healthy human serum PCR product); 3-8: PCR products from a portion of the patient sample.
FIG. 3 is an electrophoretogram of PCR amplification products obtained in step 6 from a part of patient samples in example 3. M: marker, the strip size is from top to bottom in proper order: 8000bp, 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100 bp; 1-8: PCR products of part of the samples, 9, 10: negative control (pEASY-T1 vector plasmid DNA).
FIG. 4 shows the results of clinical tests (HBsAg118K/145A variation) performed on hepatitis B patients by the gene chip.
FIG. 5 shows the result of clinical examination of hepatitis B patient by gene chip (wild type HBsAg 126T).
FIG. 6 shows the results of clinical tests (HBsAg 120Q/126I/145R variants) performed on hepatitis B patients by the gene chip.
FIG. 7 shows the results of clinical tests on the gene chip on hepatitis B patients (wild type HBsAg120P, 126T, 145G).
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1 preparation of Gene chip
Based on the prevalence characteristics of Hepatitis B Virus (HBV) sequences in our country, oligonucleotide probes for detecting 4 HBsAg variant sites were designed, as shown in Table 1 (the probes in Table 1 all performed NH at the 3' end2Modified).
The wild type of the HBsAg is shown as a sequence 14 in a sequence table. The 4 HBsAg variation sites are respectively: (1) s118: HBsAg amino acid 118; (2) s120: HBsAg amino acid 120; (3) s126: HBsAg amino acid 126; (4) s145: HBsAg amino acid 145.
TABLE 1 Probe information
| Name of probe | Probe sequence (5 '-3') | Number of bases |
| 118T19 | CTACCAGCACGGGACMATGTTTTTTTTTTTT (sequence 1) | 31 |
| 118K19 | CTACCAGCAAGGGACMATGTTTTTTTTTTTT (sequence 2) | 31 |
| 120P17 | CAMGGGACCATGCAAAATTTTTTTTTTTT (sequence 3) | 29 |
| 120Q17 | CAMGGGACAATGCAAAATTTTTTTTTTTT (sequence 4) | 29 |
| 126T19 | CCTGCACGACTCCTGCTCATTTTTTTTTTTT (sequence 5) | 31 |
| 126I19 | CCTGCACGATTCYTGCTCATTTTTTTTTTTT (sequence 6) | 31 |
| 145G19 | CTTCGGACGGAAACTGCACTTTTTTTTTTTT (sequence 7) | 31 |
| 145A19 | CTTCGGACGCAAACTGCACTTTTTTTTTTTT (sequence 8) | 31 |
| 145R19 | CCTTCGGACAGAAACTGCATTTTTTTTTTTT (SEQ ID NO: 9) | 31 |
After synthesizing 9 probes shown in Table 1, each probe was dissolved in a spotting solution (3 XSSC, 0.01% SDS) at a concentration of 100. mu.M. And (3) using a spotting instrument to spot a probe on the surface of the aldehyde glass sheet, and drying overnight to prepare the gene chip, wherein the distribution diagram of the gene chip is shown in figure 1 (P is a quality control probe, and N is a negative probe).
Quality control probe: 5'-TTTTTTTTTTTTTTTTTTTT-3', biotin labeling at the 5 'end and NH at the 3' end2And (5) modifying.
Negative probe: 5'-CAAGCTGACTCTAGCAGATCTTTC-3' (plant gene sequence unrelated to hepatitis B virus).
After the gene chip is used for detection, the quality control probe hole is positive, and the negative probe hole is negative, so that the result is effective. The detection result reading method comprises the following steps:
a 118T19 probe well positive indicates that the 118 th amino acid of the HBsAg is wild type (T), and a 118K19 probe well positive indicates that the 118 th amino acid of the HBsAg is mutant type (K);
a positive 120P17 probe well indicates that the 120 th amino acid of the HBsAg is wild type (P), and a positive 120Q17 probe well indicates that the 120 th amino acid of the HBsAg is mutant type (Q);
a 126T19 probe hole positive indicates that the 126 th amino acid of the HBsAg is wild type (T), and a 126I19 probe hole positive indicates that the 126 th amino acid of the HBsAg is mutant type (I);
a probe hole of 145G19 positive indicates that the amino acid at position 145 of the HBsAg is wild type (G), a probe hole of 145A19 positive indicates that the amino acid at position 145 of the HBsAg is mutant type (A), and a probe hole of 145R19 positive indicates that the amino acid at position 145 of the HBsAg is mutant type (R).
T is threonine, K is lysine, P is proline, Q is glutamine, I is isoleucine, G is glycine, A is alanine, and R is arginine.
Example 2 establishment of detection method
1. And (4) taking serum to be detected, and extracting virus DNA by using a virus DNA out kit according to a method of a specification.
2. Taking the virus DNA obtained in the step 1 as a template, and carrying out nested PCR reaction to obtain a PCR amplification product (about 1200 bp);
the method comprises the following specific steps:
(1) first round PCR, reaction system: 5. mu.l of template (viral DNA), 12. mu.l of PCR reaction solution, 2.5. mu.l of each of upstream and downstream primers, ddH2O2.5. mu.l, Taq enzyme 0.5. mu.l; PCR reaction procedure: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 35s, annealing at 59 ℃ for 35s, extension at 72 ℃ for 70s, and 2 cycles; denaturation at 94 ℃ for 35s, annealing at 57 ℃ for 35s, extension at 72 ℃ for 70s, 2 cycles; denaturation at 94 ℃ for 35s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 70s for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 53 ℃ for 35s, and extension at 72 ℃ for 70s for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 51 ℃ for 35s, and extension at 72 ℃ for 70s, for 2 cycles; denaturation at 94 ℃ for 35s, annealing at 55 ℃ for 35s, and extension at 72 ℃ for 70s for 30 cycles; extension at 72 ℃ for 7 min.
Upstream primer (SEQ ID NO: 15 of sequence Listing): 5'-AGTCAGGAAGACAGCCTACTCC-3', respectively;
downstream primer (sequence 16 of sequence listing): 5'-AGGTGAAGCGAAGTGCACAC-3' are provided.
(2) Second round PCR, reaction system: 5. mu.l of template (first round PCR product), 15. mu.l of PCR reaction solution, 0.5. mu.l of each of upstream and downstream primers, ddH2O28.5 mul, Taq enzyme 0.5 mul; PCR reaction procedure: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 25s, annealing at 59 ℃ for 25s, extension at 72 ℃ for 50s, and 30 cycles; extension at 72 ℃ for 7min and at 68 ℃ for 5 min.
Upstream primer (SEQ ID NO: 17 of sequence Listing): 5'-TTCCTGCTGGTGGCTCCAGTTC-3', respectively;
downstream primer (SEQ ID NO: 18 of the sequence Listing): 5'-TTCCGCAGTATGGATCGGCAG-3' are provided.
3. And (3) purifying the PCR amplification product obtained in the step (2) by adopting an ethanol precipitation method, and then adding A tail at the tail end of the purified product by adopting Taq enzyme.
4. Connecting the product processed in the step 3 to a pEASY-T1 vector, and carrying out colony PCR for positive colony identification (the positive PCR amplification product is about 300 bp); and (3) PCR reaction system: single colony, PCR reaction solution 12. mu.l, upstream and downstream primers 0.25. mu.l, ddH2O12.4. mu.l, Taq enzyme 0.1. mu.l; PCR reaction procedure: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 25s, annealing at 56 ℃ for 25s, extension at 72 ℃ for 50s, and 35 cycles; extension at 72 ℃ for 10 min.
An upstream primer: 5'-TTCCTGCTGGTGGCTCCAGTTC-3', respectively;
a downstream primer: 5'-TTCCGCAGTATGGATCGGCAG-3' are provided.
5. And (4) taking the positive colony identified in the step 4 to culture and extracting plasmid DNA.
6. And (3) performing PCR amplification by using the plasmid DNA obtained in the step (5) as a template, wherein a PCR reaction system comprises: template (plasmid DNA) 5. mu.l, PCR reactionSolution 12. mu.l, primer F2200.25. mu.l, primer R5501.25. mu.l, ddH2O6. mu.l, Taq enzyme 0.5. mu.l; PCR reaction procedure: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 5min after 35 cycles.
Primer F220 (sequence 10): 5'-TGGATGTGTCTGCGGCGTT-3', respectively;
primer R550 (sequence 11): 5'-CGAACCACTGAACAAATGGCAC-3' are provided.
The 5' end of primer R550 was biotinylated.
7. And (3) thermally denaturing the product obtained in the step (6) at 98 ℃ for 5min, then quickly moving the product to an ice bath, and standing the product for 5 min.
8. Mu.l of the product treated in step 7 and 5. mu.l of a hybridization solution (2 XSSC, 1.2% SDS, 5% formamide) were mixed, added to the spotting area of the gene chip prepared in example 1, placed in a hybridization cassette to hybridize in a 45 ℃ water bath for 1 hour, then washed sequentially with solutions A (1 XSSC, 0.2% SDS), B (0.2 XSSC) and C (0.1 XSSC) for 20 seconds, spun-dried, 10. mu.l of a labeling solution (horseradish peroxidase-labeled streptavidin) was added to the spotting area, placed in a hybridization cassette in a 37 ℃ water bath for 25 minutes, the chip was removed, washed with 1 XPBST (1 XPBS + 0.05% Tween 20) for 20 seconds, and spun-dried. And adding equal volumes of chromogenic reagent A liquid and chromogenic reagent B liquid into each chip reaction area in a dark place, putting the chip into a biochip imager for scanning, and judging the HBsAg mutation site condition according to the scanning result.
In the actual detection, the steps 3 to 5 may be omitted, and the PCR amplification product obtained in step 2 may be directly taken as a template and subjected to the operations of steps 6, 7 and 8.
Example 3 clinical sample testing
Study subjects: 40 chronic hepatitis B patients, 24 men and 16 women, which accord with the division of the Chinese medical society hepatopathy and the division of the Chinese medical society infectious etiology: the chronic hepatitis B prevention and treatment guideline (2015 edition) is the diagnosis standard of chronic hepatitis B, and can be used for eliminating autoimmune diseases and other liver diseases.
Serum samples were taken from the subjects and tested as described in example 2.
The electrophoresis chart of the PCR amplification product obtained by part of samples through the step 2 is shown in figure 2, and the sizes of the amplification products are all about 1200bp and are consistent with the expectation; the electrophoresis chart of the PCR amplification product obtained in step 6 is shown in FIG. 3, and the sizes of the amplification products are all about 300bp, which is consistent with the expectation.
The gene chip prepared by the invention detects 40 cases of chronic hepatitis B patients confirmed by clinical diagnosis. Chip detection results show that 3 cases of T118K/G145A mutation, 2 cases of T126I/G145R mutation, 2 cases of P120Q/T126I/G145R mutation, 2 cases of T126I/G145R mutation, 26 cases of T126I mutation, 1 case of T126I/G145A mutation, 1 case of G145R mutation and 3 cases of no mutation in 40 patients. The results of the partial detection are shown in FIGS. 4 to 7. FIG. 4 shows the results of the detection of 1 HBsAg118K/145A mutation in patients. FIG. 5 shows the results of 1 example 126T (non-mutated) patient. FIG. 6 shows the test results of 1 case of P120Q/T126I/G145R mutant patients. FIG. 7 shows the results of 1 example of 120P/126T/145G (non-mutated) patients.
And comparing the detection result with the DNA sequencing result, wherein the result shows that the result is completely consistent with the DNA sequencing result when the gene chip is used for detection.
Claims (10)
1. A set of probes consisting of probe P1, probe P2, probe P3, probe P4, probe P5, probe P6, probe P7, probe P8 and probe P9;
the probe P1 is a single-stranded DNA molecule shown in sequence 1 of the sequence table;
the probe P2 is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;
the probe P3 is a single-stranded DNA molecule shown in sequence 3 of the sequence table;
the probe P4 is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;
the probe P5 is a single-stranded DNA molecule shown in a sequence 5 in a sequence table;
the probe P6 is a single-stranded DNA molecule shown in sequence 6 of the sequence table;
the probe P7 is a single-stranded DNA molecule shown in a sequence 7 in a sequence table;
the probe P8 is a single-stranded DNA molecule shown in a sequence 8 in a sequence table;
the probe P9 is a single-stranded DNA molecule shown in a sequence 9 of a sequence table.
2. The use of the kit of probes of claim 1, which is (b1) or (b 2):
(b1) detecting HBsAg site mutation in a sample to be detected;
(b2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
3. A gene chip obtained by immobilizing the set of probes according to claim 1 on a chip medium.
4. The use of the gene chip of claim 3, which is (c1) or (c 2):
(c1) detecting HBsAg site mutation in a sample to be detected;
(c2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
5. A primer chip combination consisting of the gene chip of claim 3 and a primer pair;
the primer pair consists of a primer F and a primer R;
the primer F is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 11 of a sequence table.
6. A primer probe combination consisting of the set of probes and primer pairs of claim 1;
the primer pair consists of a primer F and a primer R;
the primer F is a single-stranded DNA molecule shown as a sequence 10 in a sequence table;
the primer R is a single-stranded DNA molecule shown in a sequence 11 of a sequence table.
7. The primer chip combination of claim 5 or the primer probe combination of claim 6, wherein the primer chip combination is (e1) or (e 2):
(e1) detecting HBsAg site mutation in a sample to be detected;
(e2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
8. A kit A containing the probe set of claim 1 or a kit B containing the gene chip of claim 3 or a kit C containing the primer chip combination of claim 5 or a kit D containing the primer probe combination of claim 6; the application of the kit A or the kit B or the kit C or the kit D is (f1) or (f 2):
(f1) detecting HBsAg site mutation in a sample to be detected;
(f2) preparing a kit for detecting HBsAg site mutation in a sample to be detected.
9. A method for detecting HBsAg site mutation in a sample to be detected comprises the following steps: carrying out PCR amplification by using nucleic acid of an isolated sample to be detected as a template and adopting the primer pair as claimed in claim 5 to obtain a PCR amplification product; hybridizing the PCR amplification product with the gene chip of claim 3, and judging HBsAg site mutation in a sample to be detected according to a hybridization result.
10. The method of claim 9, wherein: the hybridization comprises the following steps: (1) hybridizing the PCR product with the gene chip at 45 ℃ for 1 h; (2) horse radish peroxidase labeled streptavidin was added to the chip and hybridized in a 37 ℃ water bath for 25 min.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1431317A (en) * | 2002-10-14 | 2003-07-23 | 山东省医药生物技术研究中心 | Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus |
| CN101008031A (en) * | 2006-01-24 | 2007-08-01 | 中国药品生物制品检定所 | Mutation checking gene chip for hepatitis B virus 'a' determinant |
| CN101182585A (en) * | 2007-12-12 | 2008-05-21 | 博奥生物有限公司 | Method for identifying HBV gene mutation type, special chip and reagent kit |
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| US6759193B2 (en) * | 1999-07-28 | 2004-07-06 | The Government Of The Republic Of Singapore | Detection of human hepatitis B virus surface antigen mutants by specific amplification and its application on gene chip |
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| CN1431317A (en) * | 2002-10-14 | 2003-07-23 | 山东省医药生物技术研究中心 | Oligonucleotide chip and its application of detecting muatatonal site of hepatitis B virus |
| CN101008031A (en) * | 2006-01-24 | 2007-08-01 | 中国药品生物制品检定所 | Mutation checking gene chip for hepatitis B virus 'a' determinant |
| CN101182585A (en) * | 2007-12-12 | 2008-05-21 | 博奥生物有限公司 | Method for identifying HBV gene mutation type, special chip and reagent kit |
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