CN102230025A - Kit and method for detecting drug resistance mutation of hepatitis B virus genes - Google Patents
Kit and method for detecting drug resistance mutation of hepatitis B virus genes Download PDFInfo
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Abstract
The invention discloses a kit and method for detecting drug resistance mutation of hepatitis B virus genes. The kit comprises a primer pair for PCR (polymerase chain reaction) amplification of the hepatitis B virus gene P domain, a solid phase detection array coated with drug resistance detection probes and contrast probes on the surface, a nucleic acid amplification reaction system containing DNA polymerase and reaction substrates and loaded on a solid phase carrier, and color-producing reagents. Based on the gene chip technique in combination with the PCR technique, the detection method provided by the invention has high throughout capacity and high specificity and is rapid and easy to promote. The detection probes are immobilized on the detection array and hybridized with the detected gene fragments which are amplified by PCR, the nucleic acid sequence is synthesized and extended after the target fragment is identified by the probes on the solid phase carrier, so that the biotin-labeled amplified fragment is immobilized on the detection array instead of being eluted, and then the fragment is visualized to indicate the drug resistance mutation situation of hepatitis B viruses required to be detected.
Description
Technical field
The invention belongs to field of medical examination, relate to the sudden change Drug Resistance Detection of hepatitis B virogene, particularly a kind of hepatitis B virogene medicament-resistant mutation detection kit and detection method of being used for.
Background technology
The whole world has 1/3rd population to infect hepatitis B virus (HBV) approximately.In recent years, along with the enforcement of hepatitis B vaccine plan, this serology chronic infection mark recall rate of worldwide hepatitis B surface antigen obviously reduced.But at present 3.5 hundred million people being arranged approximately still is the chronic HBV infection person, and these patients have the risk of suffering from chronic hepatopathy, liver cirrhosis and liver cancer.Antiviral therapy is the unique selection of control and prevention chronic hbv-infection progress.
At present, there is the granted chronic hepatitis B that is used for the treatment of of multiple medicine in China.In broad terms, these medicines can be divided into two classes: cytokine and nucleotide analog.Interferon, rabbit is a kind of abiogenous cytokine, has antiviral and immunoregulatory effect, also is the medicine that first kind of approval is used for the treatment of chronic hepatitis B.Interferon, rabbit is effective to about 1/3rd patient, and needs to be undertaken by subcutaneous injection, because a lot of untoward reactions is arranged, needs to observe during treating.Nucleoside analog is an effective inhibitors at hepatitis B virus reversed transcriptive enzyme and hepatitis B virus duplication.Their advantage is oral medicine and relative less untoward reaction.Yet they must take just tool curative effect for a long time, and chemical sproof is a main aspect that limits its prolonged application.The mechanism and the timely and effective Studies on Diagnosis that produce about resistance seem particularly important for rational medicinal design and resistance patient's management.
For example, lamivudine has extraordinary antiviral therapy effect as a line medicine of treatment hepatitis B.But along with the prolongation of treatment time, under the selection pressure of antiviral therapy medicine, the wild strain of medicaments insensitive is suppressed, and persister is because of to the insensitive advantage virus strain that becomes of medicine.For example take during the lamivudine therapy, tyrosine-methionine(Met)-aspartic acid-aspartic acid (YMDD) gene order variation of archaeal dna polymerase appears under selection pressure, make 204 methionine(Met) (M) of its coding sport Xie Ansuan (V) or Isoleucine (I), i.e. YVDD that often says and YIDD variation.These two kinds of variations all cause the avidity of lamivudine and HBV archaeal dna polymerase to descend or disappear, and HBV reduces thousands of times to its susceptibility.If untimely diagnosis is also carried out rational therapy, will cause the failure for the treatment of.
Yet the real-time fluorescence quantitative PCR detection technique of having only the YMDD medicament-resistant mutation at present is applied to the clinical qualitative detection of carrying out, this method is less owing to designing complexity and flux to Drug Resistance Detection, and rely on costliness together, can only under the better laboratory condition, finish at present, not fast, accurately, the appropriate method to hepatitis B virogene sudden change Drug Resistance Detection of big flux, the early treatment of hepatitis B has been caused delay to a certain extent.
Summary of the invention
The problem that the present invention solves is to provide a kind of hepatitis B virogene medicament-resistant mutation detection kit and detection method of being used for, and is that the basis is carried out flux and detected with the gene chip, has realized high specific and fast the sudden change of hepatitis B virus is detected.
The present invention is achieved through the following technical solutions:
A kind ofly be used for the test kit that the hepatitis B virogene medicament-resistant mutation detects, comprise be used for P district primer that pcr amplification hepatitis B virogene medicament-resistant mutation detects, surface is fixed with the Drug Resistance Detection probe and contrast probe the solid phase detection arrays, comprise nucleic acid amplification reaction system and colouring reagents on the solid phase carrier of archaeal dna polymerase and reaction substrate;
Described Drug Resistance Detection probe comprises the Drug Resistance Detection probe at one or more resistance of hepatitis B virus medicine sudden changes, its length is 15～25nt, wherein be arranged on after the Drug Resistance Detection probe 5` end 5nt, and reduce sterically hindered sequence at Drug Resistance Detection probe 5` end and prolong and modify and/or auxiliary fixing the modification at detecting the mutational site;
Described colouring reagents comprises the compatible reaction liquid that contains avidin-alkaline phosphatase and contains the color reaction liquid of NBT/BCIP.
Described P district primer is no more than 1000bp to the fragment length that is increased; On detection probes, also add the modification that PNA, LNA or sulfenyl improve detection specificity.
Described Drug Resistance Detection probe comprises following one or more detection probes:
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of lamivudine resistance sudden change detection site rtL180M shown in SEQ ID NO:1;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of lamivudine resistance sudden change detection site rtM204V/I shown in SEQ ID NO:2;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of adefovir ester medicament-resistant mutation detection site rtA181V/T shown in SEQ ID NO:3;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of adefovir ester medicament-resistant mutation detection site rtN236T shown in SEQ ID NO:4;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtI169T shown in SEQ ID NO:5;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtS202I shown in SEQ ID NO:6;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtM250V/I shown in SEQ ID NO:7.
Described contrast probe comprises negative control probe and positive control probe, and its designed sequence does not all have complementarity with sequence to be detected, and the 5` end of positive control probe is also by biotin labeling.
Describedly reduce sterically hindered sequence and prolong that to modify be that prolongation in that Drug Resistance Detection probe 5` end carries out polyT, polyC, polyT+polyC or spacer 6～15C sequence is modified;
The carrier of described solid phase detection arrays is nitrocellulose filter, nylon membrane or slide glass, and auxiliary fixedly being modified at Drug Resistance Detection probe 5` least significant end of Drug Resistance Detection probe 5` end added amino, sulfydryl, hydroxyl or aldehyde radical.
Described compatible reaction liquid consists of 1mL ddH
2O and 2 μ l avidin-alkaline phosphatase liquid; Color reaction liquid consists of 1mL colour developing damping fluid, 6.5 μ lNBT and 3.5 μ l BCIP.
A kind of resistance of hepatitis B virus medicine sudden change detection method based on the mentioned reagent box may further comprise the steps:
1) is template with the DNA that comprises hepatitis B virogene group to be measured, to being amplimer, carries out a plurality of round-robin pcr amplifications, obtain pcr amplification product with primer;
2) pcr amplification product is carried out denaturing treatment after, place nucleic acid amplification reaction system on the solid phase carrier, and then add the solid phase detection arrays, carry out the solid-phase nucleic acid amplification reaction; Its response procedures is: A: 10s at least anneals under the temperature that is not less than 5 ℃ of probe Tm values; B:72 ℃ is extended 10s at least; The A-B cycle number is at least greater than 1; Last 72 ℃ are extended 3min at least;
3) after solid-phase nucleic acid amplification is finished, take out the solid phase detection arrays and put into the centrifugal wash-out of elutriant, the combination of removing non-specific nucleic acid with compatible reaction liquid covering solid phase detection arrays and hatch, is used ddH behind the wash-out once more
2The O flushing covers the color reaction liquid that comprises NBT and BCIP at last and develops the color, and uses ddH after seeing the colour developing spot
2The O termination reaction;
6) colour developing spot pairing detected result is the hepatitis B virogene wild-type detection site that is detected and does not undergo mutation, according to colour developing spot and sudden change detection probes institute at gene mutation site, judge the hepatitis B virus situation of suddenling change.
Described solid phase detection arrays before use also volumetric concentration be among 2%～5% the BSA 37 ℃ seal 15min at least.
Described denaturing treatment is that thermally denature is handled: place 5min more than 95 ℃ at least, place 3min at least at 0 ℃ again;
Perhaps, described denaturing treatment is that alkaline denaturation is handled: the NaOH solution of mass concentration 10% is mixed according to 1: 10～20 volume ratio with pcr amplification product, place 3min at least.
After the reaction of described solid-phase nucleic acid amplification is finished, take out the solid phase detection arrays and at first put into elutriant A, rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Then the solid phase detection arrays is covered behind the compatible reaction liquid 37 ℃ hatch at least 10min after, put into elutriant A, elutriant B more successively, rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Take out solid phase detection arrays ddH
2O flushing covers color reaction liquid at last and develops the color, to seeing termination reaction behind the colour developing spot;
The Tris-Cl that consists of 100mmol/L pH=7.5 of described elutriant A, 300mmol/L NaCl and 4mmol/L Mg
2Cl; The Tris-Cl that consists of 100mmol/L pH=9.5 of elutriant B, 300mmol/L NaCl and 4mmol/L Mg
2Cl.
Compared with prior art, the present invention has following beneficial technical effects:
1, provided by the inventionly be used for resistance of hepatitis B virus medicine sudden change and detect, be based on the detection in Gene Mutation of nucleic acid amplification on the solid phase carrier, detection probes is fixed on the solid phase detection arrays, hybridize with the testing gene fragment of pcr amplification, after the detected probe identification of the fragment of amplification, can carry out the prolongation (solid-phase nucleic acid amplification) of sequence, make and to be fixed on the membrane array by biotin labeled amplified fragments and not by wash-out, and the sequence that can not be discerned by probe, just can not carry out the prolongation of sequence, can not be fixed by detection probes, and then be washed away; Like this by designing the distribution that a plurality of detection probes are matrix form, by once just detecting the emergent properties of gene locus to be detected.
The present invention is by based on gene chip, PCR reaction technology being the high-throughput, high specific of principle, fast and the detection method that is easy to promote, by on the solid-phase media that with the nitrocellulose filter is representative, carrying out the nucleic acid amplification reaction that specific probe causes, in conjunction with reverse dot blot hybridization technique, after will carrying biotin labeled target sequence and probe on the membrane array combining, by biotin-avidin-alkaline phosphatase-BCIP/NBT reaction, can observe obvious bluish voilet spot, thereby carry out the naked eyes interpretation.
Because the medicament-resistant mutation kind of hepatitis B virus is more, the sudden change with correspondence is relatively more difficult in conjunction with the probe design and the judgement of dyeing one by one with it, and complex operation, is easy to make mistakes; Therefore,,,, sample to be tested then develops the color accordingly as the colour developing contrast with wild-type if not undergoing mutation in the site of being detected; So, it is detected as the colour developing contrast with a plurality of wild-types, just can detect it whether medicament-resistant mutation has taken place.
The invention provides 7 kinds of Drug Resistance Detection probes, when detecting, have only a kind of probe pin colour developing in the middle of the array, carry out result's indication, other probes there is no colour developing in the membrane array; This shows that these several medicament-resistant mutation detection probes can specificly identify the mutation type of hepatitis B gene group to be measured, and its result accurately and reliably, amixia dyeing each other.
2, the detection of resistance of hepatitis B virus medicine sudden change provided by the invention, the detection accuracy height: comprise by specific probe design, utilize ripe round pcr annealing temperature to stop non-specific combination and to utilize Taq enzyme 5`-3` 5 prime excision enzyme activity to stop extension after the mispairing, can reach good discrimination power fully, ensure the accuracy that detects nucleotide sequence list base difference;
And less demanding to laboratory and plant and instrument: on common lab PCR instrument, can finish integral experiment, need not special and expensive device; Simple to operate; Mainly operating with the round pcr is core, and routine operation is with low cost, is easy to promote.
3, the detection of resistance of hepatitis B virus medicine sudden change provided by the invention is carried out at hepatitis B virogene P district, because according to reporting in the past that the sudden change in this district mainly caused the generation of hepatitis B virus drug resistance.This method detected result is reliable, accurate; And slow, the technical problems such as resolving power is not high, necessary instrument is expensive, testing process complexity of the detection speed that has solved existing method, can improve the existing detection of seldom considering before the hepatitis B patient treatment drug resistant gene significantly, and then ignore the gene resistance and cause the present situation of delay treatment.
4, the detection of resistance of hepatitis B virus medicine provided by the invention sudden change, the medicament-resistant mutation detection probes that is provided can flexible combination, to adapt to different detection crowd and testing goal, help the flexible Application of Clinical Laboratory.
Description of drawings
The program synoptic diagram that Fig. 1 combines, extends, develops the color for amplified fragments and Drug Resistance Detection probe on being fixed on solid phase carrier; Wherein 1. being nitrocellulose filter, for the 5` end prolongs the C10 chain of modifying, 3. is the Drug Resistance Detection probe 2., 4. is amplified fragments target complement sequence district, 5. is the Taq enzyme, 6. is biotin labeling, 7. is avidin-alkaline phosphatase, 8. is the BCIP/NBT colour developing;
Fig. 2 is the detection specificity authentication chip array distribution synoptic diagram of resistance of hepatitis B virus medicine sudden change;
Fig. 3 is that the specificity of resistance of hepatitis B virus medicine mutant probe is identified colour developing figure as a result; Fig. 3 a～Fig. 3 g is respectively the rtL180M that detects, rtM204V/I, rtA181V/T, the colour developing result of rtN236T, rtI169, rtS202I, rtM250V/I.
Embodiment
Detect the design of segmental amplification and detection probes in the detection below in conjunction with concrete resistance of hepatitis B virus medicine sudden change, fixing of detection probes and solid phase carrier, detection probes and amplified fragments are hybridized on solid phase carrier, are increased, the present invention is described in further detail, and the explanation of the invention is not limited.
Referring to Fig. 1, detection method of gene mutation based on nucleic acid amplification on the solid phase carrier, a kind of detection kit that is used for resistance of hepatitis B virus medicine sudden change that is provided, comprise the primer that is used for pcr amplification hepatitis B virogene P district, surface is fixed with the Drug Resistance Detection probe and contrast probe the solid phase detection arrays, comprise nucleic acid amplification reaction system and colouring reagents on the solid phase carrier of archaeal dna polymerase and reaction substrate.
According to the hepatitis B medicament-resistant mutation sequence of bibliographical information with P district gene regions as detecting the medicament-resistant mutation section, common PCR amplification in vitro is adopted in its amplification, require cycle number 25～40, the length suggestion of amplified fragments is no more than 1000bp, and amplimer both can also can specifically have been decided in the position of amplified fragments relation by mutational site to be measured on reverse primer at forward primer the mark of last avidin; The Design of length of detection probes is 15～25nt, is arranged on after the 5` end 5nt at the site of detecting mutation polymorphism on the detection probes.
Described medicament-resistant mutation detection probes comprises that its length is 15～25nt at one or more resistance of hepatitis B virus medicine sudden change detection probes, and the specific sequence site of the site of underscore mark for the wild-type of detection arranged in the middle of following sequence;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of lamivudine resistance sudden change detection site rtL180M shown in SEQ ID NO:1;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of lamivudine resistance sudden change detection site rtM204V/I shown in SEQ ID NO:2;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of adefovir ester medicament-resistant mutation detection site rtA181V/T shown in SEQ ID NO:3;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of adefovir ester medicament-resistant mutation detection site rtN236T shown in SEQ ID NO:4;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtI169T shown in SEQ ID NO:5;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtS202I shown in SEQ ID NO:6;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtM250V/I shown in SEQ ID NO:7.
And add the modification of polyT (10)+polyC (10) at its 5` end.
In the medicament-resistant mutation detection probes that is provided, each catastrophe point detection probes can be used separately, is fixed on the same distribution sites after also several probes can being mixed.
The contrast probe comprises negative control probe and positive control probe, and the sequences Design of negative control probe and positive control probe can be identical or inequality, and principle is that its sequence can not have cross complementary with amplified fragments.Whether the 5` end of positive control probe can be developed the color by biotin labeling, normal in order to the monitoring Color Appearance System; Negative control probe is in order to the specificity of monitoring hybridization and solid-phase nucleic acid amplification.
The sequence of concrete contrast probe can be designed as:
Positive control sequence B iotin-acagcactaa ggcaagctat tctg;
Negative control sequence acagcactaa ggcaagctat tctg.
The modification of Drug Resistance Detection probe 5` end comprises: reduce sterically hindered sequence and prolong modification and/or auxiliary fixing the modification; Wherein reducing sterically hindered sequence prolongation modification is for the sequence after reducing the nucleic acid amplification mispairing and increasing and the consideration of adhering to, developing the color of solid phase carrier, comprises the prolongation modification of multiple sequences such as polyT, polyC, polyT+polyC or spacer 6～15C.
5` end auxiliary fixing modifies general and solid phase carrier is taken into consideration, solid phase carrier can be nitrocellulose filter, nylon membrane or slide glass, fix with it by auxiliary fixing modification of adding amino, sulfydryl, hydroxyl or aldehyde radical at the 5` of detection probes end, the better fixing and color developing effect in the future for detection probes, solid phase carrier can carry out kinds of surface and modify.
Such as, be solid phase carrier with the nitrocellulose filter, after drying again by immersion in SSC solution, can be so that the indiffusion of colour developing spot; By the hydroxyl of detection probes 5` end, behind ultraviolet lighting, fix with nitrocellulose filter.
Solid-phase nucleic acid amplification is that detection probes and target gene fragment are hybridized on solid phase carrier, increased, it is the core of probe specificity identification, wherein, amplification system adopts conventional PCR reaction solution, its unique distinction just is with the Drug Resistance Detection probe that comprises detection site as primer, can and carry out nucleic acid amplification with the complementary hybridization of goal gene, and be fixed on the solid phase carrier not by wash-out by detection probes; Simultaneously, for the specificity that guarantees to hybridize, the annealing temperature operated by rotary motion is in being not less than 5 ℃ of scopes of probe Tm value.
Elution process is in order will not wash away with solid phase carrier bonded amplified fragments, and elutriant can be for general damping fluid commonly used, and is relatively good in conjunction with centrifugal elute effect.
Colour developing it is advantageous that by the display mode of avidin-alkaline phosphatase liquid+NBT/BCIP background color is not obvious, and reaction is accurate, and the result judges that easily effect is relatively good; And realize that conveniently avidin-alkaline phosphatase liquid and NBT/BCIP colour developing liquid all can adopt existing test kit.
Below in conjunction with the gene Drug Resistance Detection process of hepatitis B virus, test kit and detection method thereof are carried out specific description:
1) detects the preparation of using amplified production
Design of amplification primers is right, and the 5` end of reverse amplimer is increased vitamin H (Biotin) mark, and designed primer is specially P:
Forward primer: tgttcctgcg gtaaagta 18;
Reverse primer: Biotin-tgtattccca tcccatca 18;
The wild-type sequence of 7 detection site of synthetic is used for the target sequence of Drug Resistance Detection, total length 332bp, DNA with these 7 kinds of wild-type sequences is a template, is that primer carries out 30 round-robin pcr amplifications with primer to P, and can obtain the length that 7 kinds of detection site suddenly change respectively is the amplified fragments of 332bp.
2) design of detection probes is with synthetic
Design and synthetic the detection probes in 7 Drug Resistance Detection sites of hepatitis B viruses (HBV), probe Tm value is designed to 45 ± 1 ℃, probe length is 13～15nt, holds at probe sequence 5` simultaneously to add polyT (T
10), polyC (C
10) sequence prolong to modify, reducing that sterically hindered prolongation modifies is to combine with being fixed on epilamellar probe in order to be beneficial to target sequence, concrete detection probes sequence is shown in SEQ ID NO:1～SEQ ID NO:7;
The site of detecting polymorphism on the probe is provided with all after probe 5` end 5nt.
3) detection probes fixing on detection arrays
With nitrocellulose filter at 2 * SSC solution (trisodium citrate 15mM, sodium-chlor 150mM, pH7.0) take out after soaking 30min in, oven dry is 2 hours in 37 ℃ of incubators, again Drug Resistance Detection probe and contrast probe are sprayed into (1 500nl on the film with some film instrument, interval 1mm, be matrix distribution), reduce to 4mm * 6mm size, the nitrocellulose filter of having put probe was put into 80 ℃ of oven for drying 30 minutes, 254nm wavelength ultraviolet lighting 6 minutes is fixedlyed connected probe by ultraviolet lighting with nitrocellulose filter, be fixed the solid phase detection arrays of detection probes and contrast probe.Under drying conditions, the solid phase detection arrays can be laid in stand-by for a long time, before using this film is immersed 2%BSA liquid and seals 15min for 37 ℃.
The detection arrays design distributes as Fig. 2:
3.～6. the 2. negative contrast probe of the wherein 1. positive contrast probe of Fig. 2 a is rtL180M, rtM204V/I, rtA181V/T, rtN236T.
3.～5. the 2. negative contrast probe of the wherein 1. positive contrast probe of Fig. 2 b is rtI169, rtS202I, rtM250V/I.
4) solid-phase nucleic acid amplification reaction
At first the gene amplification product that the first step is obtained carries out the sequence number mark, then amplified production is carried out thermally denature and handles: 95 ℃ of water-bath 5min, place 3min then on ice; Again with the 10 μ l of the amplified production after the sex change, PCR reaction solution (glad hundred promise companies, TagMixMaster) 20 μ l, ddH
2O 10 μ l put into clean PCR reaction tubes mixing, and the detection arrays for preparing is put into, and the film back side tube wall of solid phase detection arrays carries out the solid-phase nucleic acid amplification reaction; Design response procedures: A:45 ℃ of annealing 30s; B:72 ℃ is extended 30s; The A-B cycle number is 10; Last 72 ℃ are extended 3min.
Wherein detected object is 7 kinds of sites P district gene order of not suddenling change of amplification.
5) wash-out and colour developing
After solid phase PCR reaction is finished, take out detection arrays and carry out wash-out, will not wash away with detection arrays fixed fragment, all elution process is all finished under 37 ℃ of conditions, is specially:
Detection arrays is taken out, put into elutriant A (Tris-Cl of 100mmol/L pH=7.5,300mmol/L NaCl and 4mmol/L Mg
2Cl) centrifugal wash-out in (rotating speed is 30rpm, wash-out 1min) is after the taking-up detection arrays, with compatible reaction liquid (the 1mL ddH of alkaline phosphate ester enzyme labelling
2O and 2 μ l avidin-alkaline phosphatase liquid, available from green skies company, alkaline phosphatase is labeled as Streptavidin) cover after, hatch 10min under 37 ℃ of conditions; Hatch and after finishing detection arrays is put into clean elutriant A and carry out wash-out (rotating speed 30rpm, wash-out 1min), put into elutriant B (Tris-Cl of 100mmol/L pH=9.5,300mmol/L NaCl and 4mmol/L Mg after the taking-up again
2Cl) in, centrifugal again wash-out (rotating speed 30rpm, wash-out 1min); Take out detection arrays, with clear water to the surface washing of film piece, and utilize thieving paper to remove liquid on the film piece, the film piece is covered color reaction liquid (1mL develop the color damping fluid, 6.5 μ lNBT and 3.5 μ l BCIP, available from green skies company, BCIP/NBT alkaline phosphatase colouring reagents box), after naked eyes can be seen spot after 30 seconds, promptly use the clear water stopped reaction.
6) mutation detecting analysis
Resistance of hepatitis B virus medicine sudden change detect colour developing as a result figure as shown in Figure 3, wherein 1. positive contrast, 2. negative contrast can be seen by all normally colour developings of biotin labeled positive control, illustrates that Color Appearance System is working properly; Negative control does not develop the color, and illustrates that the specificity of solid-phase amplification is good, does not have non-specific hybridization and nucleic acid amplification.All have only 3. among Fig. 3 a～Fig. 3 d, 4., 5., all have only 3. in the site in the middle of 6., Fig. 3 e～Fig. 3 g, 4., a site in the middle of 5., in institute's fixed detection probes respectively with corresponding HBV gene do not suddenly change the target sequence specific combination and the colour developing, have only a kind of probe the colour developing of medicament-resistant mutation hepatitis B virus not to take place in the middle of the array to be measured certain, carry out result's indication, other probes there is no colour developing in the membrane array; This shows that these seven kinds of medicament-resistant mutation detection probes can specificly identify the not mutation type of hepatitis B gene group to be measured, and is corresponding in case sudden change does not promptly produce spot, and its result accurately and reliably, amixia dyeing each other.
Detected result in conjunction with above array, after HBV medicament-resistant mutation detection probes provided by the present invention is fixed and forms the detection arrays of array distribution, every kind of detection probes all can be carried out result's indication at certain medicament-resistant mutation hepatitis B virus colour developing to be measured, its colour developing obviously, the result accurately and reliably.
Accuracy and repeatability textual criticism:
To four kinds of HBV medicament-resistant mutation type rtL180M, rtM204V/I, rtA181V/T, each 25 routine Chinese resistance of hepatitis B virus medicine patient's of rtN236T serum HBV amplified production checks order, compare with above-mentioned method detected result, it is 99% that this method detects accuracy rate again.Wherein 2 routine flase drops cause because of producing sudden change in the next door, site of designing probe base.
Claims (10)
1. one kind is used for resistance of hepatitis B virus medicine sudden change detection kit, it is characterized in that, comprise be used for P district primer that pcr amplification hepatitis B virogene medicament-resistant mutation detects, surface is fixed with the Drug Resistance Detection probe and contrast probe the solid phase detection arrays, comprise nucleic acid amplification reaction system and colouring reagents on the solid phase carrier of archaeal dna polymerase and reaction substrate;
Primer right 5` end in described P district increases biotin labeling;
Described Drug Resistance Detection probe comprises the Drug Resistance Detection probe at one or more resistance of hepatitis B virus medicine sudden changes, its length is 15～25nt, wherein be arranged on after the Drug Resistance Detection probe 5` end 5nt, and reduce sterically hindered sequence at Drug Resistance Detection probe 5` end and prolong and modify and/or auxiliary fixing the modification at detecting the mutational site;
Described colouring reagents comprises the compatible reaction liquid that contains avidin-alkaline phosphatase and contains the color reaction liquid of NBT/BCIP.
2. the resistance of hepatitis B virus medicine sudden change detection kit that is used for as claimed in claim 1 is characterized in that described P district primer is no more than 1000bp to the fragment length that is increased; On detection probes, also add the modification that PNA, LNA or sulfenyl improve detection specificity.
3. the resistance of hepatitis B virus medicine sudden change detection kit that is used for as claimed in claim 1 is characterized in that described Drug Resistance Detection probe comprises following one or more detection probes:
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of lamivudine resistance sudden change detection site rtL180M shown in SEQ ID NO:1;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of lamivudine resistance sudden change detection site rtM204V/I shown in SEQ ID NO:2;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of adefovir ester medicament-resistant mutation detection site rtA181V/T shown in SEQ ID NO:3;
At the sequence of the Drug Resistance Detection probe of the hepatitis B virus of adefovir ester medicament-resistant mutation detection site rtN236T shown in SEQ ID NO:4;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtI169T shown in SEQ ID NO:5;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtS202I shown in SEQ ID NO:6;
At the Drug Resistance Detection probe sequence of the hepatitis B virus of Entecavir medicament-resistant mutation detection site rtM250V/I shown in SEQ ID NO:7.
4. the resistance of hepatitis B virus medicine sudden change detection kit that is used for as claimed in claim 1, it is characterized in that, described contrast probe comprises negative control probe and positive control probe, its designed sequence does not all have complementarity with sequence to be detected, and the 5` end of positive control probe is also by biotin labeling.
5. the resistance of hepatitis B virus medicine sudden change detection kit that is used for as claimed in claim 1, it is characterized in that, describedly reduce sterically hindered sequence and prolong that to modify be that prolongation in that Drug Resistance Detection probe 5` end carries out polyT, polyC, polyT+polyC or spacer 6～15C sequence is modified;
The carrier of described solid phase detection arrays is nitrocellulose filter, nylon membrane or slide glass, and auxiliary fixedly being modified at Drug Resistance Detection probe 5` least significant end of Drug Resistance Detection probe 5` end added amino, sulfydryl, hydroxyl or aldehyde radical.
6. the resistance of hepatitis B virus medicine sudden change detection kit that is used for as claimed in claim 1 is characterized in that described compatible reaction liquid consists of 1mL ddH
2O and 2 μ l avidin-alkaline phosphatase liquid; Color reaction liquid consists of 1mL colour developing damping fluid, 6.5 μ lNBT and 3.5 μ l BCIP.
7. the resistance of hepatitis B virus medicine sudden change detection method based on the described detection kit of claim 1 is characterized in that, may further comprise the steps:
1) is template with the DNA that comprises hepatitis B virogene group to be measured, to being amplimer, carries out a plurality of round-robin pcr amplifications, obtain pcr amplification product with primer;
2) pcr amplification product is carried out denaturing treatment after, place nucleic acid amplification reaction system on the solid phase carrier, and then add the solid phase detection arrays, carry out the solid-phase nucleic acid amplification reaction; Its response procedures is: A: 10s at least anneals under the temperature that is not less than 5 ℃ of probe Tm values; B:72 ℃ is extended 10s at least; The A-B cycle number is at least greater than 1; Last 72 ℃ are extended 3min at least;
3) after solid-phase nucleic acid amplification is finished, take out the solid phase detection arrays and put into the centrifugal wash-out of elutriant, the combination of removing non-specific nucleic acid with compatible reaction liquid covering solid phase detection arrays and hatch, is used ddH behind the wash-out once more
2The O flushing covers the color reaction liquid that comprises NBT and BCIP at last and develops the color, and uses ddH after seeing the colour developing spot
2The O termination reaction;
4) colour developing spot pairing detected result is the hepatitis B virogene wild-type detection site that is detected and does not undergo mutation, according to colour developing spot and sudden change detection probes institute at gene mutation site, judge the hepatitis B virus situation of suddenling change.
8. resistance of hepatitis B virus medicine as claimed in claim 7 sudden change detection method is characterized in that, described solid phase detection arrays before use also volumetric concentration be among 2%～5% the BSA 37 ℃ seal 15min at least.
9. resistance of hepatitis B virus medicine sudden change detection method as claimed in claim 7 is characterized in that described denaturing treatment is that thermally denature is handled: place 5min more than 95 ℃ at least, place 3min at least at 0 ℃ again;
Perhaps, described denaturing treatment is that alkaline denaturation is handled: the NaOH solution of mass concentration 10% is mixed according to 1: 10～20 volume ratio with pcr amplification product, place 3min at least.
10. resistance of hepatitis B virus medicine as claimed in claim 7 sudden change detection method is characterized in that, after described solid-phase nucleic acid amplification reaction is finished, takes out the solid phase detection arrays and at first puts into elutriant A, and rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Then the solid phase detection arrays is covered behind the compatible reaction liquid 37 ℃ hatch at least 10min after, put into elutriant A, elutriant B more successively, rotating speed is the centrifugal wash-out of 30rpm 1min at least at least; Take out solid phase detection arrays ddH
2O flushing covers color reaction liquid at last and develops the color, to seeing termination reaction behind the colour developing spot;
The Tris-Cl that consists of 100mmol/L pH=7.5 of described elutriant A, 300mmol/L NaCl and 4mmol/L Mg
2Cl; The Tris-Cl that consists of 100mmol/L pH=9.5 of elutriant B, 300mmol/L NaCl and 4mmol/L Mg
2Cl.
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| CN106367528A (en) * | 2016-08-26 | 2017-02-01 | 厦门大学 | Detection method of hepatitis B virus drug-resistant mutation |
| CN111172293A (en) * | 2020-02-19 | 2020-05-19 | 武汉大学 | Kit for detecting hepatitis B virus drug-resistant gene mutation site by Sanger method and detection method |
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| 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 |
| CN101545013A (en) * | 2009-03-10 | 2009-09-30 | 上海翼和应用生物技术有限公司 | Hepatitis B virus multi-drug resistant gene locus typing detection kit |
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| 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 |
| CN101545013A (en) * | 2009-03-10 | 2009-09-30 | 上海翼和应用生物技术有限公司 | Hepatitis B virus multi-drug resistant gene locus typing detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106367528A (en) * | 2016-08-26 | 2017-02-01 | 厦门大学 | Detection method of hepatitis B virus drug-resistant mutation |
| CN111172293A (en) * | 2020-02-19 | 2020-05-19 | 武汉大学 | Kit for detecting hepatitis B virus drug-resistant gene mutation site by Sanger method and detection method |
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