CN103468829B - A kind of hepatitis B virus nucleic acid kit that detects - Google Patents
A kind of hepatitis B virus nucleic acid kit that detects Download PDFInfo
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Abstract
The present invention relates to a kind of hepatitis B virus nucleic acid kit that detects. This kit comprises sample treatment solution A, sample treatment solution B, interior mark, PCR reactant liquor A, PCR reactant liquor B, negative quality-control product, positive quality control product, quantitative reference material etc. In this kit PCR reactant liquor A, comprise a pair of oligonucleotide probe, wherein fluorescence probe sequence is: 5 '-FAM-ATCACCACCATCTGGTTTCACCCGGAC-P-3 ', quenching probes sequence is: 5 '-GAAACCAGATGGTGGTGA-Dabcyl-3 ', this probe application is carried out to qualitative and quantitative detection to the hbv nucleic acid in sample in the method for quantitative fluorescent PCR, there is high sensitivity, high specific, the feature that simple to operate, detection speed is fast. In view of above advantage, mentioned reagent box can be widely used in hepatitis b virus infected auxiliary diagnosis.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of for detection of hepatitis B virus nucleic acid kit.
Background technology
It is the one of the main reasons that causes cirrhosis and stem cell cancer that hepatitis type B virus (HBV) infects. HBVIn the infected's blood plasma (clearly), HBV nucleic acid is to understand hbv replication level, HBV Infect And Diagnose, curative effect in liverThe important indicator of monitoring and direction of medication usage, accurately sensitive quantitative detection serum HBV nucleic acid is to hepatitis BThe monitoring of result for the treatment of has meaning of crucial importance. At present, fluorescence quantitative PCR method detection HBV nucleic acid is now inspectionSurvey the most frequently used means of hepatitis B virus duplication.
Although China HBV nucleic acid quantification reagent quality obtains significant improvement, the sensitivity of most reagent, standardCompared with import high-quality reagent, still there is larger gap in exactness and repeatability etc. International mainstream HBV nucleic acid is fixedAmount reagent, as being limited to 12～20IU/ml under the detection of Roche quantitative reagent. And current main flow in China marketDomestic HBV nucleic acid quantification detects lower limit and is generally 500～1000IU/ml, can not meet clinical diagnosis demand.
Summary of the invention
For above-mentioned deficiency, the object of the present invention is to provide a kind of real-time fluorescence based on combined probe technologyRound pcr detects the kit of hbv nucleic acid.
To achieve these goals, the present invention adopts following technical scheme:
The invention provides a pair of oligonucleotide probe, its fluorescence probe sequence is: 5 '-FAM-ATCACCACCATCTGGTTTCACCCGGAC-P-3 '; Quenching probes sequence is:5′-GAAACCAGATGGTGGTGA-Dabcyl-3′。
The present invention also provides a kind of kit that detects hepatitis B virus nucleic acid, and this kit comprises and contains above-mentioned widowThe PCR reactant liquor A of nucleotide probe.
Above-mentioned PCR reactant liquor A also comprises the interior mark probe for monitoring system, and this interior mark probe sequence is: 5 '-HEX-TCCATTACACGTGAACGATTCCCCA-TAMRA-3′。
Above-mentioned PCR reactant liquor A also comprises and contains Mg2+, dATP, and dCTP, dGTP, dTTP, dUTP,Formamide, Tris-HCl, KCl, the PCR reaction buffer of Tween20; Target gene forward primer, reversePrimer; Interior target forward primer, reverse primer.
Wherein, the primer of the amplification of the target gene in described PCR reactant liquor:
Forward primer 5 '-CGTCTCGAGCAACTATACTGT-3 '
Reverse primer 5 '-CACCGGTCGTGCACAGTATC-3 '.
Wherein, the interior label primer of described monitoring system is:
Forward primer 5 '-GCCTTTCATTGGCATGACCACCTTTCAT-3 '
Reverse primer 5 '-AGCCAGTCGTCATAATCGCATT-3 '.
The above-mentioned kit for detection of hbv nucleic acid also comprises sample treatment solution A, sample treatment solutionB, interior mark, PCR reactant liquor B, negative quality-control product, positive quality control product, quantitatively reference material.
Wherein, described sample treatment liquid A is 7%PEG6000;
Sample treatment liquid B comprises NaOH, TritonX-100, NP40, Tris-HCL;
PCR reactant liquor B comprises Taq enzyme and UNG enzyme;
Negative quality-control product is normal serum;
Positive quality control product is the recombinant dna fragment containing HBV nucleic acid;
Quantitatively reference material is the recombinant dna fragment containing HBV nucleic acid;
Interior mark quality-control product is the recombinant dna fragment containing luciferase.
Another object of the present invention is the application of mentioned reagent box on fast detecting hepatitis type B virus.
Above-mentioned application, comprises the following steps:
1) carry out positive and negative quality-control product with sample treatment solution A and B and utilize paramagnetic particle method to separate sample to be testedDNA。
2) DNA of extraction is joined in the PCR reaction tube that contains PCR reactant liquor A and PCR reactant liquor B,Carry out pcr amplification, use fluorescent quantitative PCR detector to detect.
The present invention compared with prior art, has following advantages:
1) high sensitivity, minimal detectable concentration is 50IU/ml;
2) totally-enclosed reaction, extracts after viral DNA, is directly used in PCR and detects, and has avoided polluting occurring;
3) detection speed is fast, and whole process is no more than 2 hours;
4) simple to operate, controllability is strong, can carry out batch samples detection, is conducive to industrialization;
5) in PCR reaction system, be added with interior mark, there is the advantages such as the false negative of preventing.
6) be added with anti-pollution system, there is the effect of the product pollution of preventing.
Visible, this kit carries out qualitative and quantitative detection by the hbv nucleic acid in sample, due toIt has high sensitivity, high specific, the feature that simple to operate, detection speed is fast, can be widely used in B-modeThe auxiliary diagnosis of hepatites virus infections.
Brief description of the drawings
Fig. 1 shows working standard amplification curve. Wherein, (1) is each standard items amplification curve, and (2) areEach standard items linear correlation graph of a relation.
Fig. 2 shows negative quality-control product Quality Identification figure. Wherein, (1) illustrates does not have B-mode liver in testing processThe pollution of scorching viral nucleic acid, experimentation can normally increase, and in (2), mark quality-control product amplification curve, illustrates detectionSystem effectively increases.
Fig. 3 shows positive quality control product amplification curve. Diagram amplification curve is S type curve, and detection architecture is describedHbv nucleic acid has effectively increased.
Detailed description of the invention
Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention. Do not carrying on the backUnder the prerequisite of the present invention's spirit and essence, modification made for the present invention or replacement, all belong to of the present inventionCategory.
Embodiment 1: a kind of assembling that detects hepatitis B virus nucleic acid kit
Kit of the present invention is made up of following part: sample treatment solution A, sample treatment solution B, interior mark, PCRReactant liquor A, PCR reactant liquor B, negative quality-control product, positive quality control product, quantitatively reference material.
Component and the proportioning of mentioned reagent box each several part are as follows:
Sample treatment liquid A:7%PEG6000.
Sample treatment liquid B: contain NaOH, TritonX-100, NP40,1MTris-HCL (PH8.0).
PCR reactant liquor A: contain Mg2+, dATP, dCTP, dGTP, dTTP, dUTP, formamide,Tris-HCl, KCl, the PCR reaction buffer of Tween20; Target gene and interior target forward primer, reversePrimer; Oligonucleotide probe and interior mark probe, aqua sterilisa.
PCR reactant liquor B: comprise Taq enzyme and UNG enzyme;
Negative quality-control product: normal serum;
Positive quality control product: containing the recombinant dna fragment of HBV DNA;
Quantitatively reference material: containing the recombinant dna fragment of HBV DNA;
Interior mark quality-control product: containing the recombinant dna fragment of luciferase.
This kit, the amount of said components is:
Sample treatment liquid A:5.0mlx2 pipe; Sample treatment liquid B:1.3mlx2 pipe; PCR reactant liquor A:1.4mlx1Pipe; PCR reactant liquor B:60ulx1 pipe; Negative quality-control product: 400ulx1 pipe; Positive quality control product: 400ulx1Pipe; 1.～4. quantitative reference material: each 400ulx1 pipe; Interior mark quality-control product: 100ulx1 pipe.
Embodiment 2: a kind of foundation that detects hepatitis B virus nucleic acid kit method
1, sample collection
This kit is applicable to serum or plasma sample. Gather serum: get person under inspection's venous blood 2ml, be collected inIn aseptic centrifuge tube, room temperature is placed and is no more than 4 hours, and centrifugal 20 minutes of 3000rpm draws serum and proceeds toFor subsequent use in another aseptic centrifuge tube; Gather blood plasma: get person under inspection's venous blood 2ml in contain anti-coagulants aseptic fromIn core barrel, (ban use of anticoagulant heparin), room temperature is placed and is no more than 4 hours, centrifugal 20 minutes of 3000rpm,Separated plasma, proceeds to aseptic centrifuge tube for subsequent use.
2, nucleic acid extraction
1) get negative control and join in sample treatment liquid A, additional proportion is 1:49, adds as required; GetPositive control joins in sample treatment liquid A, and additional proportion is 1:49, adds as required; Get quantitative reference materialJoin in sample treatment liquid A, additional proportion is 1:49, adds as required; Stand-by.
2) draw 100 μ l sample treatment liquid A to centrifuge tube with band filter core suction nozzle, in this centrifuge tube, add100 μ l samples to be tested.
3) lid upper tube cap, vibration mixes, centrifugal 10 minutes of 13000rpm when centrifugal (fixing centrifuge tube direction).
4) inhale abandon supernatant (precipitate not obviously, need suct clearly at centrifuge tube bottom precipitation offside, should disposable suctionSupernatant to the greatest extent, avoids stirring or encountering precipitation).
5) in above-mentioned centrifuge tube, add 50 μ l sample treatment liquid B, fully vibration mixes, low speed (2000rpm)After the centrifugal several seconds, 100 DEG C dry bathes or boiling water bath 10 minutes, puts into immediately-20 DEG C of refrigerators, places 5min.
6) 1min that gently pearl suspended, centrifugal 2 minutes of 5000rpm, abandons supernatant.
7) add 50 μ lddH2O, concussion mixes, centrifugal 2 minutes of 13000rpm, getting supernatant is that PCR is anti-(supernatant, if do not used immediately, need be put-20 DEG C of preservations, needs centrifugal 1 point of 13000rpm while reusing to answer templateClock).
3, PCR reagent is prepared
1) get after PCR reactant liquor A in kit, the thawing of PCR reactant liquor B room temperature low speed (2000rpm)The centrifugal several seconds.
2), according to sample number to be amplified, every person-portion is according to the ratio preparation of PCR reactant liquor A:B=26.5:0.5PCR mixed liquor, and be filled in PCR reaction tube by 27 μ l/ pipes point, will the reaction tube of PCR mixed liquor be housedMove to sample process district.
4, application of sample
Get respectively processed good sample, negative control, positive control, working standard with the suction nozzle with filter core,Be designated as 1.～4. the each 3 μ l of supernatant are added to respectively in the PCR reaction tube that PCR reactant liquor is housed. Lid upper tube cap,After the centrifugal several seconds, move to augmentation detection district.
5, pcr amplification detects
1) PCR reaction tube being put into fluorescent PCR amplification instrument increases.
2) loop parameter is set, specifically in table 1:
Table 1: quantitative fluorescent PCR period
3) instrument sense channel is selected: FAM is HBV amplified signal, and HEX is interior mark amplified signal.
6, interpretation of result
Analyze according to the software of each quasi-instrument, obtain the HBV detection of nucleic acids result of each sample.
Threshold principle with threshold line just above normal negative control curve (random noise line)High point is as the criterion. Baseline is chosen 6～10 or 6～15 race ways.
7. result judgement
1) for 5 × 101IU/ml≤HBV nucleic acid≤5 × 108The sample of IU/ml, the corresponding knot of measuring of reportReally.
2) for HBV nucleic acid > 5 × 108The sample of IU/ml, measured result is only for reference, indicates > in report5×108IU/ml. If need accurate quantification, can, according to measured result, this sample be diluted to 5 × 10 with negative serum8Repetition measurement after IU/ml is following.
3) for 1 × 101IU/ml≤HBV nucleic acid < 5 × 101The sample of IU/ml, measured result is only for reference,In report, indicate < 5 × 101IU/ml or quantitative result are only for reference.
4) for the HBV negative sample (HBV detection of nucleic acids value is 0IU/ml) that increases, as interior mark resultThe upper < 5 × 10 that indicates of positive report1IU/ml or lower than kit detect lower limit. As negative in interior mark resultTackle this sample and carry out repetition measurement.
Embodiment 3: a kind of use that detects quality-control product in hepatitis B virus nucleic acid kit
In hbv nucleic acid detection kit, quality-control product comprises working standard, positive quality control product, feminine genderQuality-control product and interior mark, for clinical testing quality control, method of operating is with sample to be checked.
Quality control standard is as follows:
The testing result of working standard should be in quantitative scope, and the coefficient correlation of calibration curve | r| >=0.98,The results are shown in Figure 1, diagram amplification curve is S type curve, and gradient is normal, and linear dependence is good.
Negative quality-control product measurement result negative (HBV detection of nucleic acids value is 0IU/ml), and interior mark result isThe positive, the results are shown in Figure 2, and diagram amplification curve is more straight broken line, there is no intersection point with fluoroscopic examination threshold line,Curve is not S-type, and interior mark result is positive, and the dirt that there is no hbv nucleic acid in testing process is describedDye, experimentation can normally increase.
The measurement result of positive control is positive, and Ct value to be less than 35(HBVDNA detected value be 5 × 102～5×104IU/ml, the results are shown in Figure 3, and diagram amplification curve is S type curve, illustrates that detection architecture has effectively increasedHbv nucleic acid.
Above requirement must meet in same once experiment simultaneously, otherwise it is invalid that this experiment is regarded as, need again enterOK.
Embodiment 4: a kind of hepatitis B virus nucleic acid detection kit that detects is applied
Liver cancer, posthepatitic cirrhosis that this test selects doubtful Hepatitis B Virus Infection, HBV to causePatient, other hepatites virus infections person of non-hepatitis B infection, healthy population, as research object, always countBecome 204 routine effective sample tests, sample type is serum, uses existing on the market detection kit as rightContrast detection according to reagent, judge not for contrast agents box and kit testing result yin and yang attribute of the present inventionThe case of symbol, adopts goldstandard sequence measurement to check detection.
Result: the positive group of 175 example sample, the negative group of 29 example sample, kit of the present invention and the inspection of contrast agents boxSurvey result and take off blind contrast, 1 routine sample kit of the present invention and contrast agents box testing result are not inconsistent. Divide by statisticsAnalyse, two kinds of positive coincidence rates of reagent are 100%, and negative match-rate is 96.6%, and total coincidence rate is 99.5%.McNemar Chi-square Test result shows kit of the present invention and the inconsistent part of contrast agents box testing resultDifference not statistically significant (χ 2=1, P=1.000 ﹥ 0.05). Kappa consistency check result shows, needs checkingComment kit and contrast agents box testing result uniformity fabulous (Kappa=0.980, Z=13.995, P ﹤0.0002). Correlation analysis result shows that kit of the present invention and contrast agents box have significant correlation (phaseClose coefficient R2=0.9709, regression equation is: y=1.0412x-0.1734). Inconsistent to testing result1 routine sample uses goldstandard sequence measurement to check, and checks result and shows this example sample results and the present invention's examinationAgent testing result conforms to.
Above result shows that hepatitis B virus nucleic acid detection kit of the present invention (PCR fluorescence probe method) is with normalRule detection method set up kit testing result statistically without significant difference, two kinds of reagent testing resultsUniformity is fabulous, equivalence on hbv nucleic acid measuring ability. Visible, kit provided by the inventionFor hepatitis b virus infected auxiliary diagnosis.
Claims (8)
1. a pair of oligonucleotide probe, is characterized in that, fluorescence probe sequence is: 5 '-FAM-ATCACCACCATCTGGTTTCACCCGGAC-P-3 '; Quenching probes sequence is: 5 '-GAAACCAGATGGTGGTGA-Dabcyl-3 '.
2. contain the detection reagent of oligonucleotide probe claimed in claim 1.
3. detect a hepatitis B virus nucleic acid kit, it is characterized in that, comprise the PCR reactant liquor A that contains oligonucleotide probe claimed in claim 1, the primer of the target gene amplification in described PCR reactant liquor is:
Forward primer 5 '-CGTCTCGAGCAACTATACTGT-3 '
Reverse primer 5 '-CACCGGTCGTGCACAGTATC-3 '.
4. kit according to claim 3, is characterized in that, above-mentioned PCR reactant liquor A also comprises the interior mark probe for monitoring system.
5. kit according to claim 4, is characterized in that, above-mentioned interior mark probe sequence is: 5 '-HEX-TCCATTACACGTGAACGATTCCCCA-TAMRA-3 ', and the interior label primer of monitoring system is:
Forward primer 5 '-GCCTTTCATTGGCATGACCACCTTTCAT-3 '
Reverse primer 5 '-AGCCAGTCGTCATAATCGCATT-3 '.
6. according to the kit described in claim 3～5 any one, it is characterized in that, above-mentioned PCR reactant liquor A also comprises and contains Mg2+, dATP, dCTP, dGTP, dTTP, dUTP, formamide, Tris-HCl, KCl, the PCR reaction buffer of Tween-20.
7. kit according to claim 3, is characterized in that, also comprise sample treatment solution, interior mark quality-control product, the PCR reactant liquor B that comprises Taq enzyme and UNG enzyme, negative quality-control product, positive quality control product, quantitatively reference material wherein one or more.
8. kit according to claim 7, is characterized in that, described sample treatment solution comprises treatment fluid A and treatment fluid B, and wherein treatment fluid A is 7%PEG6000; Treatment fluid B comprises NaOH, TritonX-100, NP40, Tris-HCl; Negative quality-control product is normal serum; Positive quality control product is the recombinant dna fragment containing hepatitis B virus nucleic acid; Quantitatively reference material is the recombinant dna fragment containing hepatitis B virus nucleic acid; Interior mark quality-control product is the recombinant dna fragment containing luciferase.
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| CN109321678A (en) * | 2018-09-27 | 2019-02-12 | 上海裕隆神光医学检验实验室有限公司 | A kind of hepatitis B DNA detection kit containing specific PCR buffer |
| CN109694926A (en) * | 2018-12-29 | 2019-04-30 | 中山大学达安基因股份有限公司 | A kind of method and kit of quantitative detection HBV nucleic acid |
| CN113061664A (en) * | 2021-03-30 | 2021-07-02 | 百沃特(天津)生物技术有限公司 | Nucleic acid composition for detecting African horse sickness virus and kit thereof |
| CN114540542A (en) * | 2021-12-10 | 2022-05-27 | 山东博弘基因科技有限公司 | Hepatitis B virus nucleic acid detection kit with high analysis sensitivity |
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