CN101812537B - Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus - Google Patents

Abstract

The invention provides a method for simultaneously detecting three nucleotide analog drug resistance sites of hepatitis B virus and a kit thereof. Based on the genotype sequence of hepatitis B virus, the invention designed four nested amplification primers in the HBV polymerase region and twenty-seven wild-type oligonucleotide probes for drug resistance detection at ten drug resistance sites. Needle, use Digoxigenin-labeled oligonucleotide universal primers to perform nested PCR reaction to amplify the target DNA fragment, and use the labeled target DNA amplification product to hybridize with the specific oligonucleotide probe on the substrate, through hybridization The enzyme-catalyzed color reaction coupled with the conjugate can determine the existence of HBV drug resistance to the three nucleotide analogues. The method improves the accuracy, reliability, and sensitivity of detection, and can simultaneously detect 10 drug-resistant sites of three nucleoside analogs, thereby realizing high-throughput, multi-site, economical, rapid detection, and more close meet clinical needs. It is of great significance to realize early monitoring and rationally guide clinical individualized medication.

Description

Method and kit for simultaneously detecting three nucleotide analog drug resistance sites of hepatitis B virus

technical field

The invention relates to a method for detecting drug resistance of hepatitis B virus, in particular to a method for detecting drug resistance of hepatitis B virus to three types of nucleotide analogues by reverse dot hybridization technology, and also relates to a kit for clinical testing.

Background technique

Hepatitis B virus infection is widespread worldwide, especially in my country, which accounts for 1/3 of the world's infected people. 5%-10% of chronically infected patients can lead to liver cancer (HCC), 30% progress to liver cirrhosis, and 23% of patients with liver cirrhosis will develop liver failure within 5 years. According to statistics, the annual medical and health care expenses for HBV-infected patients in my country are as high as 100 billion RMB. Therefore, HBV infection seriously endangers people's health and causes serious losses to the national economy.

Anti-HBV drug therapy has been recognized internationally as the key to effective treatment. At present, there are mainly two categories of interferons and nucleoside analogues (NRTIs). Interferon mainly exerts its effects through immune regulation and anti-virus, with relatively long-lasting curative effect, less drug resistance, and more obvious adverse reactions, especially not suitable for patients with decompensated liver function. Nucleotide analogues (NRTIs) inhibit viral replication by inhibiting reverse transcriptase activity and terminating chain elongation. At present, the nucleoside analogue drugs against HBV mainly include the following three categories: L-nucleotide analogues, acyclic nucleoside phosphates, and deoxyguanosine analogues. The representative drugs are: lamivudine (lamivudine) adefovir dipivoxil and enticavir. Nucleotide analogs have strong effects and few adverse reactions, and are widely used in clinical treatment, but long-term use can easily lead to drug-resistant mutations.

HBV is a hepadnavirus with fast replication speed, and the DNA polymerase necessary for its replication lacks a strict correction mechanism, resulting in a high mutation rate of the HBV genome itself. Under stress, drug resistance mutations will also appear to varying degrees. Clinical experiments have shown that resistance to any single drug will show cross-resistance to other drugs in the family to a certain extent, which may reduce the treatment sensitivity of other families of NRTIs. Then, the emergence of drug-resistant mutant strains will cause HBV DNA replication to rebound and the activity of alanine aminotransferase ALT to increase, which will seriously affect the prognosis, and may even cause the spread of new mutant strains, bringing hidden dangers to global public health. Therefore, effective and dynamic monitoring of HBV drug resistance to different drugs can reduce the formation of drug-resistant mutants and cross-resistance between drugs, and has great clinical significance for the selection of individualized treatment options for HBV-infected patients.

Hepatitis B virus is an incomplete double-stranded DNA. Under the action of DNA polymerase, the negative strand of HBV DNA is synthesized by reverse transcription, and then the corresponding positive strand DNA is complementary synthesized to complete the replication of the HBV genome. Nucleotide analogues such as lamivudine, adefovir dipivoxil and entecavir all act on the process of reverse transcription. Among them, the reverse transcriptase active region RT of DNA polymerase is the target region of drug action, and related drug resistance mutations mainly occur in this region. At present, the methods used for the detection of drug-resistant mutations mainly include direct sequencing, restriction fragment length polymorphism (RFLP), single-strand conformational polymorphisms (single-strand conformational polymorphisms, SSCP), real-time fluorescent PCR, and based on Gene chip technology based on the principle of reverse hybridization and linear probe analysis.

(1) Direct sequencing of PCR products: It can detect almost all possible mutations in the entire sequence, but it is easy to ignore the sequences of low-abundance drug-resistant strains that are less than 20% of quasispecies, and the sequencing results are susceptible to non-specific bands, etc. , resulting in high background and inaccurate interpretation of results. Although cloning technology can make up for the above shortcomings, it needs to analyze a large number of clones, which is relatively labor-intensive.

(2) Restriction fragment length polymorphism analysis (RFLP): first design wild-type and drug-resistant specific endonuclease recognition sites for each known drug-resistant site, after PCR and enzyme digestion , according to the enzyme cleavage map to reflect whether drug resistance mutations occur. Although its detection sensitivity is high, since each detection site needs to establish an independent enzyme cleavage reaction system, the operation is cumbersome, and not every drug resistance site can find the corresponding enzyme cleavage site, which is technically difficult .

(3) Single-strand conformational polymorphism analysis (SSCP): When monitoring the emergence and dynamic changes of drug-resistant strains of viruses, mutations of single bases can be detected, but the amplified PCR products should not be too long, preferably in It is about 150bp, and if the point mutation occurs at both ends of the amplified fragment, the spatial conformation caused by the point mutation is very small, resulting in almost the same electrophoretic mobility, and false negative results are prone to occur.

(4) Real-time fluorescent PCR: detect HBV mutants with different drug resistance sites by different melting temperatures, but it is difficult to analyze multiple sites or adjacent sites.

(5) Gene chip technology: high-throughput, multi-site detection of drug-resistant mutations of HBV is possible, but the detection of amplified products requires special instruments, well-trained technicians and multi-step washing and incubation steps, and the process is complicated and time-consuming .

(6) Linear probe analysis method: follow the principle of reverse hybridization, first design a specific probe according to the sequence to be tested and solidify it on the support, perform PCR amplification on the specific sequence of the sample to be tested, and then combine the amplified product with the solid The phase probes were hybridized in reverse, and the results were obtained by color development. At present, the INNO-LiPAHBV drug-resistant mutation kit produced by Innogenetics of Belgium is often used abroad. When the drug-resistant mutant strain only accounts for a small proportion of the total virus, LiPA analysis can detect it. Therefore, when the disease progression is at a high risk This approach has advantages. But its price is expensive, and the import price of 20 test strips is 10,000 yuan, which is limited to experimental research in China.

Other genotype and mutation detection methods still under development include mass spectrometry, computer-aided array analysis, luciferase assays, or electrochemical methods based on guanine oxidation. These detection methods are fast and accurate, but require expensive instruments, high work intensity, high technical requirements for operators, and are not suitable for mass screening, and are only used for laboratory analysis. Therefore, there is an urgent need to develop new economical and convenient drug resistance mutation detection kits.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a simple, fast, sensitive, specific, high-throughput, economical and practical detection technology, and realizes the simultaneous detection of three nucleotide analog drug-resistant sites of hepatitis B virus detection. On this basis, the invention also provides a molecular kit for simultaneously detecting three nucleotide analog drug resistance sites of the hepatitis B virus.

The technical scheme that the present invention adopts is as follows:

A method for simultaneously detecting three nucleotide analog drug-resistant sites of hepatitis B virus, comprising the following steps: (1) extracting HBV DNA genome from hepatitis B virus positive serum or plasma; (2) according to domestic Drug-resistant amino acid substitutions and corresponding base mutations of three representative nucleotide analogues of lamivudine, adefovir, and entecavir reported in foreign literature, design oligonucleotide universal primers in the polymerase region and use high (3) Use labeled universal primers to amplify the target DNA fragment by PCR reaction; (4) Add multiple oligonucleotide probes Polynucleotide tails, immobilized on the substrate, followed by UV cross-linking and 80°C baking; (5) using labeled target DNA amplification products and specific oligonucleotide probes immobilized on the substrate Hybridization; (6) whether the enzyme-catalyzed reaction of alkaline phosphatase on the hybridization conjugate is colored or not, to judge the existence of HBV resistance to three nucleotide analogues; wherein the step (2 ) The designed outer primers for the digoxigenin labeling of PCR reaction amplification are:

HBV 570F 5'-TGTTGCTGTACAAAACCT-3'SEQ NO 1

HBV 1180R 5'-TCAGCAAACACTTGGCA-3' SEQ NO 2

The inner primers are:

HBV 610F 5'-TGTATTCCCATCCCATCATC-3'SEQ NO 3

HBV 998R 5'-CCCARAAGMCCCCACAATTC-3' SEQ NO 4

The probe designed in the step (2) is:

Amplification Control Probes:

HBV 690R 5'-CGAACCACTGAACAAATGG-3'SEQ NO 5

27 specific probes for the corresponding 10 drug resistance sites:

I169: 5'-CCATAGGVVATYTTGCGA-3' SEQ NO 6

169T: 5'-CCATAGGWGTYTTGCGA-3' SEQ NO 7

V173: 5'-GAGGCCCACTCCCATAG-3' SEQ NO 8

173L::5'-GAGGCCCARTCCCATAG-3'SEQ NO 9

173G: 5'-TGAGGCCCCCTCCCATA-3' SEQ NO 10

L180: 5'-CTGAGCCARGAGAAACG-3' SEQ NO 11

180M: 5'-CTGAGCCATGAGAAACG-3' SEQ NO 12

A181: 5'-AAACTGAGCCARGAGAA-3' SEQ NO 13

181T: 5'-AAACTGAGTCARGAGAA-3' SEQ NO 14

181V: 5’-TAAACTGAACCARGAGA-3’ SEQ NO 15

T184:: 5'-TGGCACTAGTAAACTGA-3' SEQ NO 16

184G: 5'-TGGCACTACCAAACTGA-3' SEQ NO 17

S202: 5'-CCATATAACTGAAAGCC-3' SEQ NO 18

202I: 5'-CCATATAAATGAAAGCC-3' SEQ NO 19

202G: 5'-CATATAACCGAAAGCCA-3' SEQ NO 20

M204: 5'-ATCATCCATATAACTGA-3' SEQ NO 21

204V: 5'-ATCATCCACATAACTGA-3'SEQ NO 22

204I: 5'-ACATCATCDATATAACT-3'SEQ NO 23

204S: 5'-CATCATCCGAATAACTG-3'SEQ NO 24

Q215: 5'-TCAAGRTGTTGYACAGA-3' SEQ NO 25

215S: 5'-TCAAGRTGTGAYACAGA-3'SEQ NO 26

N236: 5'-GGGTTYAAATGTATACC-3' SEQ NO 27

236T: 5'-GGGGTYAAATGTATACC-3'SEQ NO 28

M250: 5'-ATTACATATCCCATRAA-3'SEQ NO 29

250V: 5’-ATTACATATCCCACRAA-3’SEQ NO 30

250I: 5'-ATTACATATCCDATRAA-3'SEQ NO 31

250L: 5'-ATTACATATCCCARRAA-3'SEQ NO 32

Wherein the number of markers of the labeled primer Digoxigenin in step (2) is 3 at the 5' end.

Wherein step (3) adopts nested PCR, the annealing temperature of PCR outer amplification is 56 ℃, the annealing temperature of inner amplification is 46 ℃, the primer concentration used is 20pmol/μl, Mg ²⁺ concentration is 2mmol/L.

Wherein the substrate in step (4) is positively charged nylon membrane, nitrocellulose membrane, preferably positively charged nylon membrane.

Wherein step (4) said oligonucleotide probe plus polynucleotide tail chain is to add 20 bases T at the 5' and 3' ends of the probe.

The present invention also provides a molecular kit for simultaneous detection of three nucleotide analog drug-resistant sites of hepatitis B virus, which is characterized in that it includes primers for polymerase chain reaction whose sequence is SEQ NO 1-4, fixed There are membrane strips whose sequences are specific probes of SEQ NO 5-32, and a hybridization solution containing tetramethylammonium chloride at a concentration of 3M.

Step (6) by immobilizing multiple specific oligonucleotide probes on the same hybridization membrane, and then through hybridization, antigen-antibody binding, and enzymatic substrate color reaction, to simultaneously detect multi-site drug-resistant mutations Purpose.

The molecular kit of the present invention specifically includes: (1) primers used for polymerase chain reaction: SEQ NO 1～4 (2) hybridization reaction reagents include: prehybridization solution (6XSSC, 0.5% SDS and 100 μ g/mL salmon essence DNA), hybridization solution (6XSSC, 0.5% SDS, 100μg/mL salmon sperm DNA and 3M tetramethylammonium chloride at molar concentration), eluent 1 (2x SSC, 0.1% SDS), eluent 2 ( 0.5x SSC, 0.1% SDS), eluent 3 (washing buffer maleic acid and sodium chloride), blocking solution (0.2% skim milk powder and 0.1% SDS), digoxin antibody (conjugated alkaline phosphatase AP : purchased from Roche), chromogenic buffer (detection buffer, 0.1M Tris, 0.1M NaCl, pH9.5), chromogenic solution (NBT/BCIP stock solution: purchased from Roche), membrane strips for hybridization and standard reference substances . (3) Carrier nylon membrane strips for hybridization reactions, on which there are specific drug-resistance probes SEQ NO 6 to SEQ NO 32. In the optimization of probe and hybridization system, the present invention selects the hybridization solution that contains tetramethylammonium chloride (TMACL), and the Tm value of its hybrid has nothing to do with the base composition of the probe, only relevant to its length. In the present invention, The length of the detection probe is 17bp, which unifies the detection conditions of all probes, so that multiple drug resistance sites can be detected at the same time, which reduces the difficulty of probe design, simplifies the complexity of hybridization conditions, and improves the stability of detection sex.

The technical principle that the present invention adopts is as follows:

Drug-resistant mutations caused by nucleotide analogs mainly occur in the RT region of HBV polymerase. Therefore, firstly, universal primers labeled with digoxin were designed to amplify the entire drug-resistant region (including the three reported nucleotide analogs common drug resistance loci). Then design specific wild and drug-resistant probes for different drug-resistant mutation sites, immobilize them on the substrate, use specific probes to hybridize with PCR products, and PCR product-labeled digoxin and alkaline phosphatase Digoxigenin antibody binding is displayed by the alkaline phosphatase substrate BCIP/NBT to achieve the purpose of simultaneously detecting multi-site drug resistance mutations. In the present invention, the forward primer is labeled with Digoxigenin, and is detected by amplifying the target sequence and hybridizing with the reverse probe. Needle (that is, the sequence complementary to the reverse probe) for hybridization detection, the effect is the same.

The design of the probe in the reverse hybridization system is the key to this project, and the rationality of the probe design directly affects the sensitivity and specificity of the detection. Considering the polymorphism of hepatitis B virus variation, the present invention is based on the 981 HBV gene sequences published by GenBank, and uses MEGA software and ClustalX software to perform molecular phylogenetic tree analysis and homologous comparison respectively to obtain the gene map of A-H eight types . Design universal primers in the drug-resistant region, and design wild and drug-resistant specific probes based on the relevant drug-resistant mutation sites of three representative drugs of lamivudine, adefovir, and entecavir reported in domestic and foreign literature, including I169T , V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I/S, Q215S, N236T, M250V/I/L This design can not only cover common drug resistance mutation sites, but also detect Mixed infection of wild-type and drug-resistant mutants during treatment. In addition, aiming at the weak signal of traditional dot hybridization, the present invention adds a polynucleotide tail chain to the oligonucleotide probe to increase the strength of the hybridization signal.

Interpretation of results:

In the present invention, wild probes and drug-resistant probes at all sites are fixed on the membrane strip, and when hybridized with the amplified product of the sample to be tested, the color will develop at the corresponding drug-resistant site, thereby judging the HBV resistance to Presence and status of three classes of nucleotide analogue resistance.

(1) Color reaction control line This line is used as a color control to indicate the effectiveness of the color development process. For HBV positive samples, it should always be positive. (2) Amplification control probe line In hybridization reaction Additional controls for amplification products, used to verify efficient amplification of HBV and correct hybridization process, should always be positive for HBV positive samples.

(3) Detection of specific drug resistance sites and interpretation of corresponding drug resistance (see Figure 1 and Table 1).

Table 1 Amino acid substitutions at drug-resistant sites of three nucleotide analogues

Lamivudine Adefovir Entecavir 169 rtI169T 173 rtV173L/G 180 rtL180M 181 rtA181T/V 184 rtT184G 202 rtS202G/I 204 rtM204V/I/S 215 rtQ215S 236 rtN236T 250 rtM250V/I/L

In Figure 1 and Table 1, the probes all adopt the international general nomenclature format, with the first amino acid in the RT region as the first position, the wild amino acid is written before it, and if there is an amino acid variation, it is written after it, such as RT The 204th amino acid in the region, the wild type is written as M204 (methionine is abbreviated as M), and the resistant one is written as 204V (valine is abbreviated as V). When the wild amino acid becomes drug-resistant, there are several possible amino acid substitutions, and there are many possibilities for each amino acid to replace its corresponding codon (three bases), such as the wild-type M (methionine) at position 204, May be replaced by V (valine) or I (isoleucine) or S (serine).

The card reading step is: compare the results obtained from the test strip with the comparison card to read the corresponding sites, and then compare it with Table 1 to confirm which drug is resistant. For a sample, there may be multi-site drug resistance, and there may also be cases of multi-drug resistance.

In order to confirm the detection accuracy of the present invention, using the method and molecular kit of the present invention, the clinical DNA samples of 47 patients with chronic hepatitis B treated with nucleotide analogs were tested for drug resistance, and the results were as follows: 47 Two of the samples in the sample could not be ideally amplified because the copy number was too low, so the present invention and the direct sequencing method could not be used for detection. The remaining 45 samples were detected by the present invention, and 14 samples were wild type, and the remaining 31 samples developed resistance. There were 44 cases of drug mutations that were consistent with the results of direct sequencing, and one case could not be interpreted due to excessive background signals generated by non-specific band interference in the direct sequencing method. At the same time, the 47 samples were subjected to copy number quantitative analysis, and samples with more than 4×10 ³ copies/ml could be effectively detected.

The beneficial effects of the present invention are:

(1) The present invention is based on the genotype of the common hepatitis B virus in my country. According to domestic and foreign literature reports, the drug-resistant amino acid substitutions of three representative drugs, lamivudine, adefovir, and entecavir, and their corresponding sites Mutations, design universal primers in drug-resistant regions, and design corresponding wild and drug-resistant mutation-specific probes, to achieve the accuracy and reliability of probe design.

(2) The specific drug resistance detection sites and probes of the three nucleotide analogs involved in the present invention cover the latest research reports and confirmed common drug resistance mutations, and the coverage is wider and updated.

(3) The amplification primer of the present invention is labeled with digoxin, and its detection sensitivity is close to the detection level of isotope, but has the advantage of no radiation damage, thereby ensuring the safety, simplicity and practicability of the experiment, and it is compatible with biological It also has a lower detection background compared to the prime label. In addition, the use of multiple digoxigenin-labeled primers can increase the signal intensity and enhance the detection sensitivity and specificity.

(4) The present invention designs 27 wild drug-resistant specific probes on a test strip to achieve simultaneous detection of three representative drugs of three nucleoside analogs, lamivudine, adefovir, The purpose of entecavir drug-resistant loci is 10 loci, thereby achieving high-throughput, multi-locus, economical, rapid detection, and more in line with clinical needs. In addition, the probe is tailed with a polymer, which is beneficial to the immobilization of the probe and the stability of the detection.

(5) In the hybridization system, the present invention selects the hybridization solution containing tetramethylammonium chloride (TMACL), and the Tm value of its hybrid has nothing to do with the base composition of the probe, but only with its length. In the present invention, the detection probe The length of the needle is 17bp, and the detection conditions of all probes are unified, so that multiple drug resistance sites can be detected at the same time, the difficulty of probe design is reduced, and the stability of detection is improved.

Description of drawings

Figure 1 Comparison card for the detection of drug-resistant loci in reagent strip probes

Fig. 2 Electrophoresis results of extra-amplified fragments of target DNA

Figure 3 Electrophoresis results of amplified fragments in the target DNA

Figure 4 Drug resistance detection of two clinical cases

specific embodiment

Embodiment 1 extracts HBV DNA gene from serum to be tested or blood plasma

Take 200ul virus serum + 200ul 2x lysis buffer (0.02mol/L Tris-HCl, 0.01mol/L EDTA, 1% SDS) + 10ul proteinase K (20mg/ml), digest at 40°C for 1.5 hours. Use phenol Extract with phenol: chloroform (1:1) and chloroform once respectively. After mixing, 12000g x 3min, recover the aqueous phase, that is, the upper layer. Add 1/10 volume of 3mol/L sodium acetate and 2 times the volume of pre-cooled absolute ethanol to precipitate DNA, 12000g x 10 minutes, recover DNA, wash DNA once with 75% ethanol, discard supernatant, after drying, add Dissolve DNA in 20 μl of pure water, take 5 μl as template for PCR reaction, and store the rest at -20°C.

Example 2 Amplification of target DNA drug-resistant regions using digoxin-labeled oligonucleotide primers (nested PCR)

(1) External amplification of target DNA

Internal amplification primers:

HBV 570F 5'-TGTTGCTGTACAAAACCT-3' (SEQ NO 1)

HBV 1180R 5'-TCAGCAAACACTTGGCA-3' (SEQ NO 2)

1. Test tube number (N): N = number of sample numbers + 1 negative control

2. PCR reaction system (TaKaRa)

Note: The whole process was operated on ice, and the negative control was supplemented by adding 5 μl of distilled water. After adding the template, mix well and centrifuge, then place it on the PCR reaction instrument.

3. PCR cycle system:

4. Electrophoresis and externally amplified PCR product: After completing the PCR cycle, immediately take 6 μL of the PCR product and add the corresponding DNA LoadingBuffer, electrophoresis on 1% agarose gel, and detect the presence or absence of the band, wherein the size of the externally amplified fragment is 610bp ( See Figure 2). No. 1 achieved amplification and can be stored at -15°C to -25°C for hybridization; No. 2 did not get enough copies of the PCR product and needed internal amplification.

(2) Internal amplification of target DNA

Internal amplification primers:

HBV 610F 5'-TGTATTCCCATCCCATCATC-3' (SEQ NO 3)

HBV 998R 5'-CCCARAAGMCCCCACAATTC-3' (SEQ NO 4)

1. Test tube number (N): N = number of externally amplified sample numbers + 1 negative control

2. PCR reaction system (TaKaRa)

Note: The whole process is operated on ice, and the negative control is supplemented by adding 1 μl of distilled water. After adding the template, mix well and centrifuge, and then place it on the PCR reaction instrument.

3. PCR cycle system:

4. Internally amplified PCR product in electrophoresis: No. 2 for external amplification should be stored at -15°C to -25°C after completion of external amplification and internal amplification, or immediately take 6 μL of PCR product and add corresponding DNA Loading Buffer, and store in Electrophoresis was carried out on 1% agarose gel to detect the presence or absence of bands, and the size of the internal amplified fragment was 388bp (see Figure 3).

The preparation of embodiment 3 hybridized nylon film strips

Dilute each artificially synthesized probe to a final concentration of 10 pmol/μl, and fix it on the corresponding position of the membrane strip. The preparation of the membrane strip is as follows:

(1) Add 27 specific probes, two samples at a time to the No. 1 pump and No. 2 pump of the automatic film spraying machine. After spraying, clean the two pipes with distilled water, and then replace the other two probes. Needle spray film.

(2) Immobilizing the probe: After the probe is fixed, after the probe is naturally dried at room temperature, it is subjected to ultraviolet cross-linking, and then baked at 80° C. for 30 minutes. The oligonucleotide probe is added with a polynucleotide tail chain, and 20 bases T are added to both the 5' and 3' ends of the probe.

(3) Cutting: After fixing, cut the film into 3mm×7cm film strips with a strip cutter along the marking line.

The drug resistance detection of embodiment 4 two typical clinical cases

The detection consists of 4 main steps: specimen denaturation, hybridization, band washing and color development.

Preparation: 2 clean and dry reaction tanks, preheating and pre-hybridization in a water bath, hybridization solution, eluent at 2-44°C, open two constant temperature shaking boxes to 25°C and 44°C, 100°C boiling water, filled with Box of ice cubes.

(1) Pre-hybridization: Add 2ml of preheated pre-hybridization solution to the hybridization tank, immerse the prepared nylon membrane strips in it, and then place it in a 44°C constant temperature shaking box (70rpm) and incubate for 20min. Membrane strips can be used for hybridization immediately, or rinsed in distilled water, dried and stored in a humid environment at 4°C for one week.

(1) Specimen denaturation: Boil and denature 10 μl of the PCR product of the sample to be tested (per reaction tank) at 100°C for 5 minutes, and quickly place it on ice to cool for 5 minutes.

(2) Specimen hybridization: add 2ml of preheated hybridization solution to each reaction tank, then add 10μ denatured specimens into the tank, shake gently to mix well, so that the detection side of the detection band is completely immersed in the hybridization solution, and the reaction tank Place on a constant temperature shaker (70rpm) at 44°C and incubate for 60min to achieve hybridization.

(3) Rinse the strips: Aspirate the hybridization solution in the reaction tank, quickly add 2ml of eluent 1 to each reaction tank, and place it on a constant temperature oscillator (70rpm) at 25°C for elution for 1min; suction After the reaction solution in the tank, add 2ml of preheated eluent 2 to each reaction tank, and elute in a constant temperature oscillator (70rpm) at 44°C for 10min; suck up eluent 2, and inject 2ml of eluent 3. Elute with a constant temperature shaker (70 rpm) at 25°C for 1 min.

Preparation: prepare antibody binding solution (digoxigenin antibody 0.2μl + blocking solution 2ml)

(4) Antibody binding: Aspirate the liquid in the reaction tank, add 2ml of freshly prepared antibody binding solution, and incubate at 25°C with a constant temperature shaker (70rpm) for 30 minutes. After aspirating the reaction solution in the tank, add 2ml of eluent 3, place it on a constant temperature oscillator (70rpm) at 25°C for elution for 1min, and repeat this step once. Aspirate the liquid in the reaction tank. Add 2ml of chromogenic buffer and elute with a constant temperature shaker (70rpm) at 25°C for 1min.

Preparation: Chromogenic working solution Configuration: Add 20μl NBT/BCIP Stock Solution per ml of chromogenic buffer

(5) Color development: suck out the liquid in the reaction tank, add 2ml of color development solution, and develop color in the dark for 30 minutes. After the color development is completed, suck out the liquid in each reaction tank, inject 2ml of distilled water into each reaction tank, stop the color development, use tweezers to pick up the detection band from the hybridization tank, put the detection side up on the water-absorbing filter paper, wait for the strip Read the results after the tape is completely dry (see Figure 4 for the test results), and keep it away from light.

From left to right is the comparison card, case 1 (wild type), case 2 is a patient after entecavir treatment, the test results show that there are drug resistance mutations at 180, 202 and 204, and the mutation forms are 180M, 202G , 204V).

sequence listing

<160>32

<210>1

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as primers for PCR amplification

<400>1

5'-TGTTGCTGTA CAAAACCT-3'18

<210>2

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as primers for PCR amplification

<400>2

5'-TCAGCAAACA CTTGGCA-3'17

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as primers for PCR amplification

<400>3

5'-TGTATTCCCA TCCCATCATC-3'20

<210>4

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, and the merged base M can be A or C, according to the specific HBV genome

Sequence design, used as primers for PCR amplification

<400>4

5’-CCCARAAGMCCCACAATTC-3’19

<210>5

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>5

5'-CGAACCACTG AACAAATGG-3'19

<210>6

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base W can be A or T, and the merged base Y can be C or T, according to the HBV genome

Specific sequence design for use as hybridization probes

<400>6

5’-CCATAGGWA TYTTGCGA-3’17

<210>7

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base W can be A or T, and the merged base Y can be C or T. According to the characteristics of the HBV genome

Sequence design for use as hybridization probes

<400>7

5'-CCATAGGWG T YTTGCGA-3'17

<210>8

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>8

5'-GAGGCCCACT CCCATAG-3'17

<210>9

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>9

5'-GAGGCCCART CCCATAG-3'17

<210>10

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>10

5'-TGAGGCCCCC TCCCATA-3'17

<210>11

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>11

5'-CTGAGCCARG AGAAACG-3'17

<210>12

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>12

5'-CTGAGCCATG AGAAACG-3'17

<210>13

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>13

5'-AAACTGAGCC ARGAGAA-3'17

<210>14

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>14

5'-AAACTGAGTC ARGAGAA-3'17

<210>15

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>15

5’-TAAACTGAAC CARGAGA-3’17

<210>16

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>16

5'-TGGCACTAGT AAACTGA-3'17

<210>17

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>17

5'-TGGCACTACC AAACTGA-3'17

<210>18

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>18

5'-CCATATAACT GAAAGCC-3'17

<210>19

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>19

5'-CCATATAAAT GAAAGCC-3'17

<210>20

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>20

5'-CATATAACCG AAAGCCA-3'17

<210>21

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>21

5’-ATCATCCATA TAACTGA-3’17

<210>22

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>22

5'-ATCATCCACA TAACTGA-3'17

<210>23

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base D can be A, G or T, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

Needle

<400>23

5’-ACATCATCDA TATAACT-3’17

<210>24

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> Designed according to the specific sequence of HBV genome, used as a hybridization probe

<400>24

5’-CATCATCCGA ATAACTG-3’17

<210>25

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, and the merged base Y can be C or T. According to the characteristics of HBV genome

Sequence design for use as hybridization probes

<400>25

5’-TCAAGRTGTT GYACAGA-3’17

<210>26

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, and the merged base Y can be C or T. According to the characteristics of HBV genome

Sequence design for use as hybridization probes

<400>26

5'-TCAAGRTGTG AYACAGA-3'17

<210>27

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base Y can be C or T, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>27

5'-GGGTTYAAAT GTATACC-3'17

<210>28

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base Y can be C or T, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>28

5'-GGGGTYAAAT GTATACC-3'17

<210>29

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>29

5'-ATTACATATC CCATRAA-3'17

<210>30

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>30

5’-ATTACATATC CCACRAA-3’17

<210>31

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base D can be A or G or T, and the merged base R can be A or G, according to the HBV genome

Specific sequence design for use as hybridization probes

<400>31

5'-ATTACATATC CDATRAA-3'17

<210>32

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> The merged base R can be A or G, designed according to the specific sequence of the HBV genome, and used as a hybridization probe

<400>32

5’-ATTACATATC CCARRAA-3’17

Claims (3)

1. one kind is used for the primer that PCR reacts the digoxigenin labeled of amplification in detecting three kinds of nucleotide analog resistances of hepatitis B virus site, it is characterized in that:

Outer primer is:

HBV?570F 5’-TGTTGCTGTACAAAACCT-3’SEQ?NO?1

HBV?1180R 5’-TCAGCAAACACTTGGCA-3’SEQ?NO?2

Inner primer is:

HBV?610F 5’-TGTATTCCCATCCCATCATC-3’SEQ?NO?3

HBV?998R 5’-CCCARAAGMCCCACAATTC-3’SEQ?NO?4。

2. the primer that in detecting three kinds of nucleotide analog resistances of hepatitis B virus site, is used for the digoxigenin labeled of PCR reaction amplification as claimed in claim 1, the reference numerals that it is characterized in that digoxin is 3 at a 5 ' end.

3. a molecular agents box that detects three kinds of nucleotide analog resistances of hepatitis B virus site simultaneously is characterized in that comprising that (1) is used for the primer of the digoxigenin labeled of PCR reaction amplification, and sequence is SEQ NO 1～4; (2) hybridization reagent comprises prehybridization solution: 6 * SSC, 0.5%SDS and 100 μ G/ML salmon sperm DNAs; Hybridization solution: 6 * SSC, 0.5%SDS, 100 μ G/ML salmon sperm DNAs and volumetric molar concentration are the tetramethyl ammonium chlorides of 3M; Elutriant 1:2 * SSC, 0.1%SDS, elutriant 2:0.5 * SSC, 0.1%SDS, elutriant 3: toxilic acid and sodium-chlor; Confining liquid: 0.2% skim-milk and 0.1%SDS; The DigiTAb of coupling SEAP, colour developing damping fluid: 0.1M Tris, 0.1M NaCl, the colour developing damping fluid of pH9.5, colour developing liquid: NBT/BCIP; (3) hybridization is with film bar and standard reference material, and point has amplification control probe SEQ NO 5, colour developing control probe SEQ NO 3 and 27 specific probe SEQ NO 6～32 to corresponding 10 resistance sites on the film bar.

Images