CN101812537B

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

Info

Publication number: CN101812537B
Application number: CN2009101912390A
Authority: CN
Inventors: 黄爱龙; 张文露; 胡源; 赖国旗; 刘彦辰; 赵丽
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2009-10-27
Filing date: 2009-10-27
Publication date: 2012-08-08
Anticipated expiration: 2029-10-27
Also published as: CN101812537A

Abstract

The invention provides a method and a reagent kit for simultaneously detecting the resistance sites of the three nucleotide analogues of a hepatitis B virus (HBV). Based on the genotype sequence of the HBV, four nested amplification primers and twenty-seven wild resistance-detection oligonucleotides probes aiming at ten resistance sites are designed in an HBV polymerase area, digoxin-labeled oligonucleotides universal primers are used for conducting nested PRC reaction to amplify a target DNA segment, a target DNA amplification product to be labeled is used for hybridizing with specific oligonucleotide probes on matrix, and the existence of the resistance of the HBV to the three nucleotide analogues is judged through enzymatic color development reaction link-coupled by hybridization conjugates. The method improves the accuracy, the reliability and the sensitivity and can simultaneously detect the ten resistance sites of the three nucleotide analogues, thereby realizing the high-throughput, multi-site, economic and rapid detection and fitting to clinical needs in a better way. The method is of great significance to the realization of early detection and the proper guide of clinical personalized medication.

Description

Detect the method and the test kit thereof in three kinds of nucleotide analog resistances of hepatitis B virus site simultaneously

Technical field

The present invention relates to detect the method for resistance of hepatitis B virus medicine, particularly adopt reverse dot blot hybridization technique to detect hepatitis B virus, also relate to the test kit that is used for Clinical Laboratory three types of drug-fast methods of nucleotide analog.

Background technology

The infection of hepatitis B virus is world wide and is widely current, and especially China the infected accounts for 1/3 of the whole world.Among the chronic infection person 5%～10% can cause liver cancer (HCC), and 30% progress is liver cirrhosis, and 23% in the liver cirrhosis patient can in 5 years liver failure take place.According to statistics, China's medical treatment and health subsidies of being used for the HBV infected patient every year are used up to 1,000 hundred million Renminbi.Therefore, HBV infects the serious harm people ' s health, causes the national economy heavy losses.

The anti-hepatic-B virus medicine treatment has been known as the key of effective treatment in the world.At present, mainly contain two big types of interferons and nucleoside analogs (NRTI).Mainly through immunomodulatory and antiviral playing a role, curative effect is lasting relatively for interferons, and resistance is few, and untoward reaction is more obvious, especially is not suitable for liver function and loses compensatory patient.And nucleotide analog (NRTI) extends the effect that suppresses virus replication that reaches through suppressing reverse transcriptase activity and terminating chain.At present; The nucleoside analog medicine of anti-HBV mainly contains following 3 types: left-handed nucleotide analog; Acyclic nucleotide phosphoric acid salt, on behalf of medicine, the pancreatic desoxyribonuclease analogue be respectively: lamivudine (lamivudine) Adefovir dibasic acid esters (adefovirdipivoxil) Entecavir (enticavir).The effect of nucleotide analog is stronger, and untoward reaction is few, is widely used in clinical treatment, but life-time service very easily causes medicament-resistant mutation.

HBV is a hepadnavirus, and reproduction speed is fast, and it duplicates the rectification mechanism that necessary archaeal dna polymerase lacks strictness; Make that HBV genome self aberration rate is higher, simultaneously, along with the prolongation of treatment time; Under the selective pressure of body and medicine, medicament-resistant mutation in various degree also can appear.Clinical experiment shows that the resistance that any single medicine produces can show the cross resistance to this family's other drug to a certain extent, thereby possibly reduce the NRTI of other families treatment susceptibility.So, the appearance of resistance variant will cause the knock-on of HBV dna replication dna, gpt ALT is active raises, and has a strong impact on prognosis, even possibly cause the propagation of novel mutant strain, brings hidden danger for global public health.Therefore, effectively, can reduce the formation of medicament-resistant mutation strain and the crossing drug resistant between medicine, simultaneously HBV the infected's individualized treatment Scheme Selection had great clinical meaning with the resistance situation of dynamic monitoring HBV to different pharmaceutical.

Hepatitis B virus is incomplete double-stranded DNA, under the effect of archaeal dna polymerase, and the minus strand of the synthetic HBV DNA of rt, complementary more synthetic corresponding positive chain DNA is accomplished the HBV genome duplication.And lamivudine, nucleotide analogs such as adefovir ester and Entecavir all act on this process of this rt.Wherein the reverse transcriptase activity region R T of archaeal dna polymerase is the drug target zone, and relevant medicament-resistant mutation also mainly occurs in this zone.At present; The method that is used for the medicament-resistant mutation detection mainly comprises direct order-checking, restriction fragment length polymorphism analysis (RFLP), single-strand conformation polymorphism analysis (single-strand conformational polymorphisms; SSCP), real-time fluorescence PCR, and based on the biochip technology of reverse hybridized principle and linear probe analytical method etc.

(1) PCR product direct sequencing: can detect the nearly all possible sudden change in the whole sequence; But ignore 20% low abundance persister sequence easily less than quasispecies; And sequencing result is vulnerable to the influence of non-specific band etc., thereby produces high background, makes that interpretation is inaccurate as a result.Though clone technology can remedy above-mentioned deficiency, need to analyze a large amount of clone strains, relatively the labor intensive material resources.

(2) restriction fragment length polymorphism analysis (RFLP):, after PCR and enzyme are cut, react according to restriction enzyme mapping whether medicament-resistant mutation takes place at first to the specific nucleic acid restriction endonuclease recognition site of each known resistance site design wild-type and drug-resistant type.Though its detection sensitivity is higher, because each detection site all need be set up an independently endonuclease reaction system, complex operation, furthermore be not that corresponding restriction enzyme site can be found in each resistance site, technical difficulty is big.

(3) single-strand conformation polymorphism analysis (SSCP): when appearance of monitoring viral resistance strain and dynamic change, can detect the sudden change of single base, but amplification PCR products can not be oversize; Be preferably in about 150bp; And, if when point mutation occurs in the amplified fragments two ends, because the space conformation that point mutation causes is very little; Cause electrophoretic mobility very nearly the same, be prone to false negative result.

(4) real-time fluorescence PCR: the difference through melting temp detects the different resistance site mutation strain of HBV, but relatively more difficult for the analysis in multidigit point or adjacent site.

(5) biochip technology: can high-throughput, multidigit point detects the medicament-resistant mutation of HBV, but the detection of amplified production is needed specific apparatus and well-trained technician and multistep washing, incubation step, the process complicacy is time-consuming.

(6) linear probe analytical method: follow reverse hybridized principle; At first according to sequences Design specific probe to be measured and be solidificated on the upholder; Treat this particular sequence of mark and carry out pcr amplification, then that amplified production and solid phase probe is reverse hybridized, colour developing is obtained a result.The INNO-LiPAHBV medicament-resistant mutation test kit that the Belgian Innogenetics of external at present normal employing company produces; When causing that drug-fast mutant strain only accounts for the total virus number very during small proportion; LiPA analyzes and just can detect, and therefore this method has superiority when PD is in excessive risk.But it costs an arm and a leg, and 20 test strip import prices are 10,000 yuan, and the domestic experimental study that at present only limits to is used.

Other still comprise mass spectroscopy, area of computer aided dot-matrix analysis, luciferase analysis or based on the electrochemical means of guanine oxidation in the genotype of research and development and sudden change detection means.These detection meanss are all quick and accurate, but need expensive instrument, and working strength is big, and is high to operator's technical requirements, and is not suitable for a large amount of examinations, only is used for lab analysis.Therefore, be badly in need of the medicament-resistant mutation detection kit of development novel economizer portable type.

Summary of the invention

The present invention is directed to the deficiency of prior art, a kind of easy, quick, sensitive, special, high-throughput, economical and practical type detection technique are provided, realized simultaneously detection three kinds of nucleotide analog resistances of hepatitis B virus site.On this basis, the present invention also provides a kind of molecular agents box that detects three kinds of nucleotide analog resistances of hepatitis B virus site simultaneously.

The technical scheme that the present invention adopts is following:

A kind of method that detects three kinds of nucleotide analog resistances of hepatitis B virus site simultaneously may further comprise the steps: HBV DNA genome is extracted in (1) from hepatitis B virus male serum or blood plasma; (2) lamivudine of reporting according to domestic and foreign literature; Adefovir; The replacement of resistance amino acid and the corresponding base mutation of three kinds of nucleotide analog representative drugs of Entecavir; Also use digoxigenin labeled at polysaccharase zone design oligonucleotide universal primer, in addition wild the and medicament-resistant mutation probe of designs specificity in corresponding resistance site; (3) use the universal primer that is labeled to carry out PCR reaction amplified target dna fragmentation; (4) oligonucleotide probe adds the polynucleotide tail chain, is fixed on the matrix, fixes through UV-crosslinked and 80 ℃ of bakings successively; (5) use target DNA amplified production that is labeled and the specific oligonucleotide probe hybridization of being fixed on the matrix; Whether the enzymatic reaction of the SEAP that (6) has on the binding substances through hybridization develops the color, and judges that to three kinds of drug-fast existence of nucleotide analog whether HBV; The outer primer that wherein said step (2) designs the digoxigenin labeled that is used for PCR reaction amplification 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

Said step (2) designed probe is:

Amplification control probe:

HBV?690R 5’-CGAACCACTGAACAAATGG-3’SEQ?NO?5

Article 27, be directed against the specific probe in corresponding 10 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 reference numerals of the said labeled primer digoxin of step (2) is 3 at a 5 ' end.

Wherein step (3) adopts nest-type PRC, and the annealing temperature of the outer amplification of PCR is 56 ℃, and the annealing temperature of interior amplification is 46 ℃, and the primer concentration of use is 20pmol/ μ l, Mg ²⁺Concentration is 2mmol/L.

Wherein the said matrix of step (4) is positively charged nylon membrane, nitrocellulose filter, preferably positively charged nylon membrane.

Wherein to add the polynucleotide tail chain be that 5 ' and 3 ' end at probe all adds 20 base T to the said oligonucleotide probe of step (4).

The present invention also provides a kind of molecular agents box that detects three kinds of nucleotide analog resistances of hepatitis B virus site simultaneously, it is characterized in that comprising that sequence is the used primer of the polymerase chain reaction of SEQ NO 1～4, to be fixed with sequence be the film bar of SEQ NO 5～32 specific probes and contains the hybridization solution that concentration is the tetramethyl ammonium chloride of 3M.

Step (6) is through being fixed in many special oligonucleotide probes on the same Hybond membrane, and again through hybridization, antigen-antibody combines, and enzymatic substrate coupling reaction reaches the purpose that detects multidigit point medicament-resistant mutation simultaneously.

Specifically molecular agents box of the present invention comprises: the used primer of (1) polymerase chain reaction: SEQ NO 1～4 (2) hybridization reagent comprises: prehybridization solution (6XSSC, 0.5%SDS and 100 μ g/mL salmon sperm DNAs), hybridization solution (tetramethyl ammonium chloride of 3M when 6XSSC, 0.5%SDS, 100 μ g/mL salmon sperm DNAs and volumetric molar concentration), elutriant 1 (2x SSC, 0.1%SDS), elutriant 2 (0.5x SSC, 0.1%SDS), elutriant 3 (washing buffer toxilic acid and sodium-chlor), confining liquid (0.2% skim-milk and 0.1%SDS), DigiTAb (coupling SEAP AP: available from Roche), colour developing damping fluid (detection buffer; 0.1M Tris, 0.1M NaCl; PH9.5), the colour developing liquid (NBT/BCIP stock solution :) available from Roche, hybridization with film bar and standard reference material.(3) be used for the carrier nylon membrane bar of hybridization, point has resistance specific probe SEQ NO 6～SEQ NO32 on the film bar.In the optimization of probe and hybridization system, the present invention selects the hybridization solution that contains tetramethyl ammonium chloride (TMACL) for use, and the Tm value of its crossbred and probe based composition are irrelevant; Only relevant with its length, among the present invention, the length of detection probes all adopts 17bp; The testing conditions of having unified all probes, thus a plurality of resistances site can be detected simultaneously, reduced the difficulty of probe design; Simplify numerous and diverse property of hybridization conditions, improved the stability that detects.

The know-why that the present invention adopts is following:

The medicament-resistant mutation that nucleotide analog causes mainly occurs in HBV polysaccharase RT zone, therefore, and the design universal primer that the is marked with digoxin whole resistance zone (the common resistance site that comprises three kinds of nucleotide analogs having reported) of increasing at first.Be directed against the wild and resistance probe of different resistant mutational site designs specificity again; It is solidified on matrix; Utilize specific probe and PCR product to carry out hybridization; The digoxin of PCR product mark combines with the DigiTAb of band SEAP, and BCIP/NBT shows through alkaline phosphatase substrate, reaches the purpose that detects multidigit point medicament-resistant mutation simultaneously.Among the present invention,, detect through amplified target sequence and reverse probe hybridization to forward primer mark digoxin; Equally, also can use the digoxigenin labeled reverse primer, after the amplified target sequence; Hybridize detection with forward probe (promptly with reverse probe complementary sequence), effect is the same.

The design of reverse hybridized system middle probe is the key of this project, the whether reasonable sensitivity and the specificity that directly has influence on detection of probe design.Consider the polymorphum of hepatitis B virus variation, the present invention is the basis with 981 HBV gene orders that GenBank announces, carries out analysis of molecular evolution tree and homology contrast respectively with MEGA software, ClustalX software, obtains the gene mapping of A-H eight types.At resistance zone design universal primer; And according to the lamivudine of domestic and foreign literature report; Adefovir; Wild and the resistance specific probe of relevant resistant mutational site design of three kinds of representative drugs of Entecavir; Comprise that I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I/S, Q215S, N236T, this design of M250V/I/L not only can contain common resistant mutational site, and can detect the polyinfection of wild-type and medicament-resistant mutation type in the therapeutic process.In addition, to the more weak shortcoming of traditional dot hybridization signal, the present invention adds the polynucleotide tail chain to oligonucleotide probe, has increased intensity of hybridization signal.

Interpretation as a result:

The present invention is fixed on the wild probe and the resistance probe of all sites on the film bar, after hybridizing with the amplified production of testing sample, will develop the color in corresponding resistance site, thereby judge that HBV is to three types of drug-fast existence of nucleotide analog and situation.

(1), this line of coupling reaction wire is as the colour developing contrast; The validity of indication process color; For HBV male sample always in male (2), the amplification control line probe hybridization to the additional control of amplified production; Be used to confirm effective amplification of hepatitis B virus and correctly carrying out of crossover process, for hepatitis B virus positive male always.

(3) detection and the drug-fast interpretation of relative medicine (seeing accompanying drawing 1 and table 1) in special resistance site.

The amino acid replacement in three kinds of nucleotide analog resistances of table 1 site

	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

Among Fig. 1 and the table 1; Said probe all adopts international name form, is the 1st with first amino acid of RT district, before its wild amino acid writes on it; If amino acid variation takes place to be write on thereafter; Like the 204th amino acids in RT district, the writing M204 (methionine(Met) is abbreviated as M) of wild-type, drug-fast writing 204V (Xie Ansuan is abbreviated as V).When wild amino acid generation resistance; The replacement of its possible amino acid has several so; Each amino acid its corresponding codon of replacement (three bases) also has multiple possibility, like the wild-type M (methionine(Met)) in 204 sites, possibly be replaced by V (Xie Ansuan) or I (Isoleucine) or S (Serine).

The Card Reader step is: result and the corresponding site of comparison card contrast interpretation with test strip draws, confirm it is which kind of medicine resistance with table 1 contrast again.For a duplicate samples, possibly there is multidigit point resistance, also can there be the drug-fast situation of multiple medicine.

In order to confirm detection accuracy of the present invention; Use method of the present invention and molecular agents box, the chronic viral hepatitis B patient's of 47 parts of nucleotide analogs treatment clinical dna sample has been carried out Drug Resistance Detection, the result is following: two examples are arranged because copy number is crossed low and can't be obtained desirable amplification in the 47 routine samples; The present invention can't be used and direct sequencing detects; It is wild-type that all the other 45 examples detect 14 routine samples through the present invention, and medicament-resistant mutation has taken place all the other 31 routine samples, and consistent with direct sequencing result is 44 examples; Wherein an example can't interpretation because the interference of non-specific band has produced too much background signal in direct sequencing.Meanwhile, this 47 duplicate samples is carried out copy number quantitative analysis, 4X10 ³The above sample of copy/ml all can effectively be detected.

The invention has the beneficial effects as follows:

(1) the present invention is a support with the genotype of the common hepatitis B virus of China; According to domestic and foreign literature report lamivudine; Adefovir, the replacement of resistance amino acid and the corresponding positions point mutation thereof of three kinds of representative drugs of Entecavir are at resistance zone design universal primer; And design corresponding wild and medicament-resistant mutation specific probe, realized the accuracy and the safety of probe design.

(2) the specificity Drug Resistance Detection site and the probe thereof of three kinds of nucleotide analogs involved in the present invention have been contained current research report and certain common medicament-resistant mutation situation, and coverage rate more extensively upgrades.

(3) amplimer of the present invention adopts digoxigenin labeled; Its detection sensitivity and isotopic detection level are close, but have the advantage of "dead" injury, thereby have guaranteed security, simplicity and the practicality of experiment; And compare with biotin labeling, have lower detection background again.In addition, adopt the mode of multiple digoxigenin labeled primer, can improve strength of signal, strengthened detection sensitivity and specificity.

(4) the present invention reaches three kinds of representative drugs lamivudines that can detect three kinds of nucleoside analogs simultaneously, Adefovir through 27 wild resistance specific probes of design on a test strip; The purpose in 10 sites in the resistance site of Entecavir; Thereby realized high-throughput, multidigit point, economy; Rapid detection meets clinical needs more.In addition, probe carries out the polymer tailing, the stability that helps the fixing of probe and detect.

(5) in the hybridization system, the present invention selects the hybridization solution that contains tetramethyl ammonium chloride (TMACL) for use, and the Tm value of its crossbred and probe based composition are irrelevant; Only relevant with its length, among the present invention, the length of detection probes all adopts 17bp; The testing conditions of having unified all probes; Thereby can detect a plurality of resistances site simultaneously, reduce the difficulty of probe design, improve the stability that detects.

Description of drawings

The detection comparison card in Fig. 1 reagent strip probe resistance site

Fig. 2 target DNA fragment electrophoresis result that increases outward

Amplified fragments electrophoresis result in Fig. 3 target DNA

The Drug Resistance Detection of Fig. 4 two routine clinical cases

Specific embodiment

Embodiment 1 extracts HBV DNA gene from test serum or blood plasma

Get 200ul serum virus+200ul 2x lysis buffer (Tris-HCl of 0.02mol/L; 0.01mol/L EDTA, 1%SDS)+10ul Proteinase K (20mg/ml), put 40 ℃ of digestion 1.5 hours. with phenol and phenol: chloroform (1: 1), each extracting of chloroform are once; Behind the mixing; 12000g x 3min reclaims water, i.e. upper strata.The sodium-acetate and the absolute ethyl alcohol deposit D NA of 2 times of volume precoolings that add the 3mol/L of 1/10 volume, 12000g x 10 minutes reclaims DNA; Use 75% washing with alcohol DNA more once; Abandon supernatant, after the drying, add 20 μ l pure water dissolving DNAs; Get 5 μ l as the PCR reaction template, remaining in-20 ℃ of preservations.

Embodiment 2 uses the digoxigenin labeled Oligonucleolide primers to carry out the amplification (nest-type PRC) in target DNA resistance zone

(1) target DNA increases outward

Interior amplimer:

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

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

1, test tube numbering (N): N=sample number quantity+1 negative control

2, PCR reaction system (TaKaRa)

Attention: whole process is in operation on ice, and negative control adds 5 μ l zero(ppm) water to be supplied, add after the template mixing centrifugal after, place on the PCR reaction appearance.

3, PCR circulating system:

4, the electrophoresis PCR product that increases outward: accomplish after the PCR circulation, get 6 μ LPCR products immediately and add corresponding DNA LoadingBuffer, electrophoresis on 1% sepharose, whether the existence of test strip to be, and wherein outer amplification clip size is the 610bp (see figure 2).Realized amplification No. 1, can be stored in-15 ℃ to-25 ℃ and can be used for hybridization; The PCR product that is not enough copied for No. 2 need carry out interior amplification.

(2) amplification in the target DNA

Interior amplimer:

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

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

1, test tube numbering (N): N=sample number quantity+1 negative control that increases outward

2, PCR reaction system (TaKaRa)

Attention: whole process is in operation on ice, and negative control adds 1 μ l zero(ppm) water to be supplied, add after the template mixing centrifugal after, place on the PCR reaction appearance.

3, PCR circulating system:

4, amplification PCR product in the electrophoresis: outer No. 2 of increasing; Accomplish outer increase and interior amplification after; Should be housed in-15 ℃ to-25 ℃, perhaps get 6 μ LPCR products immediately and add corresponding DNA Loading Buffer, electrophoresis on 1% sepharose; Whether the existence of test strip, and interior amplified fragments size is the 388bp (see figure 3).

The preparation of embodiment 3 hybridization nylon membrane bars

It is 10pmol/ μ l that every probe dilution of synthetic is become final concentration, is fixed in the corresponding position of film bar, and the preparation of film bar is following:

(1) with 27 specific probes, each two samples add No. 1 pump of automatic Membrane jetter, No. 2 pumps respectively, after having sprayed, two pipelines are cleaned up with zero(ppm) water, change two probe spray films in addition again.

(2) stationary probe: stationary probe finishes, treat the probe natural drying at room temperature after, UV-crosslinked, then 80 ℃ the baking 30min.Oligonucleotide probe adds the polynucleotide tail chain, holds at 5 ' and 3 ' of probe all to add 20 base T.

(3) cut: after fixing, with slitting shear machine film is cut into the film bar of 3mmX7cm along mark line.

The Drug Resistance Detection of 4 liang of routine typical clinical cases of embodiment

Detection is made up of 4 key steps: sample sex change, hybridization, flushing band and colour developing.

Preparation work: 2 of the reactive tanks of clean dry, in water bath the preheating prehybridization, hybridization solution, is opened two constant-temperature shaking casees to 25 ℃ and 44 ℃, 100 ℃ boiling water, is filled the square box of ice cube 2～44 ℃ of elutriants.

(1) prehybridization: the prehybridization solution that adds the 2ml preheating immerses the nylon membrane bar for preparing wherein in the hybridization groove, places 44 ℃ of constant-temperature shaking casees (70rpm) again, hatches 20min.The film bar can be used for hybridization immediately, also can rinsing in zero(ppm) water, and be put in after drying in 4 ℃ of moist environments and preserve a week.

(1) sample sex change: sample P CR product 10 μ l to be checked (every reactive tank) are boiled sex change 5min for 100 ℃, put cooled on ice 5min rapidly.

(2) sample hybridization: each reactive tank adds the hybridization solution of 2ml preheating, 10 μ sex change samples is added in the groove jog mixing again; Make the detection faces of test strip upwards be immersed in the hybridization solution fully; Reactive tank is put in 44 ℃ constant temperature oscillator (70rpm), hatches 60min, realize hybridization.

(3) flushing band: aspirate the hybridization solution in the clean reactive tank, add 2ml elutriant 1 rapidly in each reactive tank, and be placed on 25 ℃ constant temperature oscillator (70rpm), wash-out 1min; In the suction socket behind the reaction solution, the elutriant 2 that adds the 2ml preheating in each reactive tank, wash-out 10min in 44 ℃ constant temperature oscillators (70rpm); Aspirate clean elutriant 2, inject constant temperature oscillator (70rpm) the wash-out 1min of 3,25 ℃ of 2ml elutriants.

Preparation work: at present prepare antibodies liquid (2 milliliters of DigiTAb 0.2 μ l+ confining liquids)

(4) antibodies: aspirate liquid in the clean reactive tank, add the freshly prepared antibodies liquid of 2ml,, hatched 30 minutes in 25 ℃ constant temperature oscillators (70rpm).Behind the reaction solution, add 2ml elutriant 3 in the suction socket, place 25 ℃ constant temperature oscillator (70rpm) wash-out 1min, repeat this step once.Liquid in the sucking-off reactive tank.Add 2ml colour developing damping fluid, 25 ℃ constant temperature oscillator (70rpm) wash-out 1min.

Preparation work: colour developing working fluid configuration: every milliliter of colour developing damping fluid adds 20 μ l NBT/BCIP Stock Solution

(5) colour developing: the liquid in the sucking-off reactive tank adds 2ml colour developing liquid, lucifuge colour developing 30min.Colour developing finishes, and the liquid in each reactive tank of sucking-off injects 2ml zero(ppm) water to each reactive tank; Color development stopping, with tweezers gripping test strip from the hybridization groove, detection faces upwards is put on the absorbent filter; Deng reading result's (detected result is seen Fig. 4) behind the band complete drying, and keep in Dark Place.

Be followed successively by comparison card from left to right, case 1 (wild-type), case 2 is the patient of Entecavir after treating, and detected result shows that 180,202 and 204 3 sites have sent out medicament-resistant mutation, and mutant form is 180M, 202G, 204V).

Sequence table

<160>32

<210>1

<211>18

<212>DNA

< 213>artificial sequence

<220>

< 223>according to the design of HBV genome specific sequence, as the primer of pcr amplification

<400>1

5’-TGTTGCTGTA?CAAAACCT-3’18

<210>2

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>2

5’-TCAGCAAACA?CTTGGCA-3’17

<210>3

<211>20

<212>DNA

< 213>artificial sequence

<220>

<400>3

5’-TGTATTCCCA?TCCCATCATC-3’20

<210>4

<211>19

<212>DNA

< 213>artificial sequence

<220>

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

Sequences Design is as the primer of pcr amplification

<400>4

5’-CCCARAAGMC?CCACAATTC-3’19

<210>5

<211>19

<212>DNA

< 213>artificial sequence

<220>

< 223>according to the design of HBV genome specific sequence, as hybridization probe

<400>5

5’-CGAACCACTG?AACAAATGG-3’19

<210>6

<211>17

<212>DNA

< 213>artificial sequence

<220>

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

The particular sequence design is as hybridization probe

<400>6

5’-CCATAGGWA?TYTTGCGA-3’17

<210>7

<211>17

<212>DNA

< 213>artificial sequence

<220>

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

The design of sequencing row is as hybridization probe

<400>7

5’-CCATAGGWG?T?YTTGCGA-3’17

<210>8

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>8

5’-GAGGCCCACT?CCCATAG-3’17

<210>9

<211>17

<212>DNA

< 213>artificial sequence

<220>

< 223>annexing base R can be A or G, according to the design of HBV genome specific sequence, as hybridization probe

<400>9

5’-GAGGCCCART?CCCATAG-3’17

<210>10

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>10

5’-TGAGGCCCCC?TCCCATA-3’17

<210>11

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>11

5’-CTGAGCCARG?AGAAACG-3’17

<210>12

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>12

5’-CTGAGCCATG?AGAAACG-3’17

<210>13

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>13

5’-AAACTGAGCC?ARGAGAA-3’17

<210>14

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>14

5’-AAACTGAGTC?ARGAGAA-3’17

<210>15

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>15

5’-TAAACTGAAC?CARGAGA-3’17

<210>16

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>16

5’-TGGCACTAGT?AAACTGA-3’17

<210>17

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>17

5’-TGGCACTACC?AAACTGA-3’17

<210>18

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>18

5’-CCATATAACT?GAAAGCC-3’17

<210>19

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>19

5’-CCATATAAAT?GAAAGCC-3’17

<210>20

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>20

5’-CATATAACCG?AAAGCCA-3’17

<210>21

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>21

5’-ATCATCCATA?TAACTGA-3’17

<210>22

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>22

5’-ATCATCCACA?TAACTGA-3’17

<210>23

<211>17

<212>DNA

< 213>artificial sequence

<220>

< 223>annexing base D can be A or G or T, according to the design of HBV genome specific sequence, visits as hybridization

<400>23

5’-ACATCATCDA?TATAACT-3’17

<210>24

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>24

5’-CATCATCCGA?ATAACTG-3’17

<210>25

<211>17

<212>DNA

< 213>artificial sequence

<220>

< 223>annexing base R can be A or G, and annexing base Y can be C or T, special according to the HBV genome

The design of sequencing row is as hybridization probe

<400>25

5’-TCAAGRTGTT?GYACAGA-3’17

<210>26

<211>17

<212>DNA

< 213>artificial sequence

<220>

The design of sequencing row is as hybridization probe

<400>26

5’-TCAAGRTGTG?AYACAGA-3’17

<210>27

<211>17

<212>DNA

< 213>artificial sequence

<220>

< 223>annexing base Y can be C or T, according to the design of HBV genome specific sequence, as hybridization probe

<400>27

5’-GGGTTYAAAT?GTATACC-3’17

<210>28

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>28

5’-GGGGTYAAAT?GTATACC-3’17

<210>29

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>29

5’-ATTACATATC?CCATRAA-3’17

<210>30

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>30

5’-ATTACATATC?CCACRAA-3’17

<210>31

<211>17

<212>DNA

< 213>artificial sequence

<220>

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

The particular sequence design is as hybridization probe

<400>31

5’-ATTACATATC?CDATRAA-3’17

<210>32

<211>17

<212>DNA

< 213>artificial sequence

<220>

<400>32

5’-ATTACATATC?CCARRAA-3’17

Claims

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.