CN103233067B - Kit and detection method for detecting hepatitis B virus core gene promoter base mutation - Google Patents
Kit and detection method for detecting hepatitis B virus core gene promoter base mutation Download PDFInfo
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Abstract
The invention discloses a kit and a detection method for detecting hepatitis B virus core gene promoter base mutation. The kit comprises a PCR reaction solution A, a PCR reaction solution B and a PCR reaction solution C. According to the present invention, the primer used by the method is designed mainly based on hepatitis B virus core gene promoter target gene and base variation thereof; competitive inhibition of the non-mutated (wild-type) reverse complementary sequence primer on the wild-type base sequence during PCR amplification is adopted to substantially prevent wild strain DNA amplification so as to accurately detect gene variation; and the kit has characteristics of convenient detection, stable result, low cost and high sensitivity.
Description
Technical field
The present invention relates to biology field, be specifically related to a kind of test kit detecting hepatitis B virus core gene promoter base mutation, also relate to a kind of detection method of hepatitis B virus core gene promoter base mutation.
Background technology
Type B viral hepatitis (HB, hePatitisB) be a kind of global communicable disease, in the chronic hepatitis B patient of nearly 3,000 ten thousand people of China, HBV variation strain great majority entrained by it show as at viral genome pre-core region Core promoter nucleotide variation, HBV BCP district variation incidence in chronic severe hepatitis B patient is high, and prompting HBV BCP variation is relevant with hepatitis B pathogenic process and coincident with severity degree of condition.
The domestic and international method for detecting transgenation has at present:
(1) PCR direct Sequencing: the direct sequence analysis that PCR primer is carried out, and be analyzed with DNA sequencing fragment, Detection Point mutable site sequence change.
(2) PCR primer restriction endonuclease analysis: the DNA fragmentation causing restriction enzyme site to change when occurring mainly for detection of point mutation, this method must relate to restriction enzyme in catastrophe point and cut when site changes and could use.
(3) PCR-oligonucleotide probe hybridization: the method utilizes oligonucleotide fragment as probe, hybridize under the prerequisite of strict control hybridization conditions, hybridization temperature requires too strict, is difficult to apply.
(4) PCR-single strand conformation polymorphism (PCR SSCP): based on the DNA single chain fragment that sequence is different, its space conformation is also different, according to the change in location of its electrophoresis, and show the difference of different sequence ss electrophoretic mobility, thus determine whether sudden change existence.
(5) specific nucleotide primer and the molecular beacon probe of synthesis is used, to 1762 and the target dna fragment of 1764, the amplification of PCR in real time, according to the amplification of PCR, analyzes and judges whether the A1762T-Gl764A sudden change of hepatitis B virus DNA in sample exists.
(6) other: as melting temperature(Tm) (Tm) detection in Gene Mutation, biochip technology etc.
The method of this research is the primer that Antisense Suppression PCR combines fluorescent probe detection hepatitis B virus core gene promoter nucleotide variation method and uses.The party's ratio juris is:
According to hepatitis B virus core gene promoter nucleotide variation design polymerase chain DNA primer, 3 ' end position of primer sequence is at mutating alkali yl, hold base sequence according to the DNA primer 3 ' of do not suddenly change (wild-type) again, hold the complementary sequence of the 1st design reverse primer reciprocal from sequence 3 '.Like this, during pcr amplification, by the competitive inhibition to wild-type base sequence, seriously block the amplification of wild strain DNA, the fluorescent probe reporter group existed in PCR reaction solution is separated also blocked, emitting fluorescence degree significantly weakens, and the base sequence amplification to variation, produce retardance hardly, there is fluorescent probe reporter group in PCR liquid can normal separation and fluorescing, therefore, when detecting same sample, by the comparison of this detected value difference, genovariation can be judged exactly.
Summary of the invention
An object of the present invention is to provide a kind of test kit detecting hepatitis B virus core gene promoter base mutation, this test kit is easy to detect, and result is stablized.
A further object of the invention there is provided a kind of method detecting hepatitis B virus core gene promoter base mutation, and the method cost is low, susceptibility is high, good stability, simple to operate.
To achieve these goals, the present invention is by the following technical solutions:
Core technology of the present invention is: according to hepatitis B virus core gene promoter target gene and nucleotide variation design polymerase chain DNA primer thereof, 3 ˊ of primer sequence hold position at mutating alkali yl, base sequence is held again according to the DNA primer 3 ' of do not suddenly change (wild-type), the 1st reverse design Primers complementary sequences reciprocal is held from sequence 3 ', during pcr amplification, by the competitive inhibition to wild-type base sequence, and seriously block the amplification of wild strain DNA, and almost retardance is not produced to the base sequence amplification of variation.When detecting same sample, setting up the PCR reaction solution not adding this complementary sequence to compare, not comparing according to the CT value difference of same Samples detection, accurately can judge hepatitis B virus core gene promoter target gene nucleotide variation.
Test kit of the present invention is for hepatitis B virus core gene promoter A1762T and the variation of Gl764A bit base.Determine that detection A1762T position and Gl764A bit base variation target gene (GENE-BANK AB198078) are: gtttaaagac tgggaggagt tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct.
Detect a test kit for hepatitis B virus core gene promoter base mutation, comprise following composition:
PCR kit A group reagent: pcr amplification can be carried out smoothly to the wild-type of hepatitis B virus core gene promoter base and saltant type, comprise following composition:
PCR reaction solution A:PCR damping fluid (1 ×), Taq DNA polymerase 1.5U, dNTP0.3 μm ol/L
HBV-A1 upstream primer 5 '-gttta aagac tggga ggagt-3 ' 0.3 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 '; 0.2 μm of ol/L
PCR kit B group reagent: pcr amplification can be carried out smoothly to the saltant type of hepatitis B virus core gene promoter A1762T base, and serious retardance is subject to its wild-type pcr amplification, comprise following composition:
PCR reaction solution B:PCR damping fluid (1 ×), Taq DNA polymerase 1.5U, dNTP0.3 μm ol/L and
HBV-B1 upstream primer 5 '-tgggg gagga gatta ggtta at-3 ' 0.3 μm of ol/L
HBV-B2 upstream antisense primer 5 '-tttaa cctaa tctcc tcccc ca-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 '; 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 '; 0.2 μm of ol/L
PCR kit C group reagent: pcr amplification can be carried out smoothly to the saltant type of hepatitis B virus core gene promoter Gl764A base, and serious retardance is subject to its wild-type pcr amplification, comprise following composition:
PCR reaction liquid C: PCR damping fluid (1 ×), Taq DNA polymerase 1-2u, dNTP0.3 μm ol/L and
HBV-C1 upstream primer 5 '-ggagg agatt aggtt aaaga-3 ' 0.3 μm of ol/L
HBV-C2 upstream antisense primer 5 '-ccttt aacct aatct cctcc-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ', 0.2 μm of ol/L
1) sample DNA process: get test serum sample 30 μ l and add equivalent DNA extraction liquid 60 μ l and mix, 95-98 DEG C of sex change in 8--9 minute, places the cooling of 0 DEG C, refrigerator 2-3 minute, 12000rpm centrifugal 5 minutes, gets supernatant liquor centrifugal for subsequent use.
2) PCR reaction solution packing: respectively get PCR reaction solution A, B and C, each 25 μ l, get Detection and Extraction HBV DNA supernatant liquor 5 μ l to be measured and add each reaction tubes.
3) pcr amplification: get point each reaction tubes installing PCR reaction solution, increases by following pcr amplification condition setting: 94 DEG C of 3 minutes denaturations, then 93 DEG C of-55 DEG C of circulations in 60 seconds in 40 seconds 40 times.
4) detected result and judgement:
(1) judgement that PCR kit A group reagent detects hepatitis B virus core gene promoter DNA is applied:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
In test kit, one group of detection reagent detected result feminine gender of hepatitis B virus core gene promoter DNA illustrates in the sample detected without hepatitis B virus DNA.The detected result positive illustrates in the sample detected to there is hepatitis B virus DNA.
(2) judgement that PCR reaction solution B group reagent detects hepatitis B virus core gene promoter A1762T nucleotide variation is applied:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
Result illustrates: positive in PCR kit A group reagent detected result, under there is hepatitis B virus DNA precondition in judgement sample, although embodiment 2 detects negative findings illustrate in the sample detected that there is hepatitis B virus DNA suddenlys change without the A1762T of hepatitis B virus DNA.The detected result positive illustrates that there is hepatitis B virus A1762T in the sample detected suddenlys change.
(3) judgement of PCR reaction liquid C group reagent hepatitis B virus core gene promoter G1764A nucleotide variation is applied:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
Result illustrates: positive in PCR kit A group reagent detected result, and under there is hepatitis B virus DNA precondition in judgement sample, although detect negative findings to illustrate in the sample detected to there is hepatitis B virus DNA, the G1764A without hepatitis B virus DNA suddenlys change.The detected result positive illustrates that there is hepatitis B virus G1764A in the sample detected suddenlys change.
Compared with prior art, the present invention has the following advantages:
1, the primer that uses of the inventive method, main dependence hepatitis B virus core gene promoter target gene and nucleotide variation base design primer thereof, and by do not suddenly change (wild-type) reverse complementary sequence primer, when pcr amplification, to the competitive inhibition of wild-type base sequence, and seriously block the amplification of wild strain DNA, accurately detect genovariation.
2, the inventive method and primer can be used for preparing hepatitis B virus core gene promoter target gene and nucleotide variation detection kit thereof.
3, according to PCR block condition, fluorescent strength determining, can relative determination hepatitis B virus core gene promoter gene and nucleotide variation degree thereof and other situation, measurement result display transgenation (positive) and do not suddenly change (feminine gender).
4. the inventive method and design of primers, accuracy for detection in Gene Mutation is very high, the shortcoming that present ordinary method (nucleotide sequencing, nucleotide sequence hybridization, PCR single stranded conformational method etc.) operation is more loaded down with trivial details, consuming time, accuracy is lower can be overcome largely, embody technical advance, accuracy, and there is practicality widely, can promote clinically.In addition because the inventive method is carried out under the best condition of pcr amplification, improve the susceptibility detecting transgenation method.
Embodiment
Unless otherwise noted, by the synthesis of Shanghai Shan Jing biotech company, the technical scheme adopted adopts conventional molecular biological technology unless otherwise noted for the primer of the present invention or nucleotide sequence.
Embodiment 1: the PCR A group detection reagent of hepatitis B virus core gene promoter DNA in test kit, to the positive quality control product of application national regulation HBV standard substance, the negative HBV serum of people, negative quality control product and different weaker concn, carry out feminine gender and the test of positive threshold value, minimal detectable concentration is test.
Pcr amplification can be carried out smoothly to the wild-type of hepatitis B virus base and saltant type, comprise following composition:
The negative HBV serum of people: without hepatitis B virus.
Negative quality control product: to make a variation wild type gene clone strain and A1762T position and Gl764A bit base anomaly gene clone bacterial strain without A1762T position and Gl764A bit base, wild type gene clone strain (bacillus coli DH 5 alpha) plasmid DNA, nucleotide sequence is:
gtttaaagac tgggaggagt tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct。
The positive quality control product of detection experiment: A1762T position and Gl764A bit base anomaly gene clone bacterial strain (bacillus coli DH 5 alpha) plasmid DNA, nucleotide sequence is:
gtttaaagac tgggaggagt tgggggagga gattaggtta atgatctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct。
* boldface letter shows variant cipher daughter nucleus nucleotide sequence.
Clone strain builds: adopt the wild-type DNA and mutant DNA and pUCm-T carrier that synthesize A1762T position and the variation of Gl764A bit base under the catalysis of ligase enzyme, build up the recombinant vectors containing object segment, and being transformed in competence DH5 α intestinal bacteria, separation and Culture obtains.
PCR reaction solution A:PCR damping fluid (1 ×), Taq DNA polymerase 1.5U, dNTP0.3 μm ol/L, and:
HBV-A1 upstream primer 5 '-gttta aagac tggga ggagt-3 ' 0.3 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 '; 0.2 μm of ol/L
Detect threshold value that is positive and feminine gender to calculate, and detect minimal detectable concentration, use hepatitis virus (HBV) the nucleic acid quantification standard substance of national regulation to do reference:
HBVDNA negative reference product (Nat'l Pharmaceutical & Biological Products Control Institute provides)
Linear sensitivity reference material (L0-L6) (Nat'l Pharmaceutical & Biological Products Control Institute provides)
Concrete implementation step:
1) sample DNA process: get negative quality control product and positive quality control product, use without the negative human serum Sample Dilution of hepatitis B virus to required concentration, get 30 μ l to add equivalent DNA extraction liquid 60 μ l and mix, 95-98 DEG C of sex change in 8--9 minute, place 0 DEG C, refrigerator cooling 2-3 minute, centrifugal 5 minutes of 12000rpm, gets supernatant liquor centrifugal for subsequent use.
2) PCR reaction solution packing: respectively get PCR reaction solution A and B, add each reaction tubes by the μ l packing of detection 1 person-portion 25 again, respectively get Detection and Extraction HBV DNA supernatant liquor 5 μ l to be measured and add each reaction tubes, every increment originally need detect with PCR reaction solution A and B two side reaction pipe simultaneously.
3) pcr amplification: get point each reaction tubes installing PCR reaction solution, increases by following pcr amplification condition setting: 94 DEG C of 3 minutes denaturations, then 93 DEG C of-55 DEG C of circulations in 60 seconds in 40 seconds 40 times.
Detected result and judgement:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
The positive quality control product of application national regulation HBV standard substance, the negative HBV serum of people and detection experiment, make the concentration samples of different dilution, carry out feminine gender and the test of positive threshold value, minimal detectable concentration is test, result is as following table, illustrate that feminine gender and Positive test threshold value are 38, minimal detectable concentration is 5.1 × 10
3iU/ml, CT are 37.52, and this susceptibility can meet hospital clinical inspection needs.
| Sample | Concentration | CT | Detected result is negative/positive |
| Negative reference product NO1 | Without HBV DNA | 40.0 | Negative |
| Negative reference product NO2 | Without HBV DNA | 38.01 | Negative |
| Negative reference product NO3 | Without HBV DNA | 38.57 | Negative |
| Negative reference product NO4 | Without HBV DNA | 40.00 | Negative |
| The negative HBV serum 1 of people | Without HBV DNA | 38.12 | Negative |
| The negative HBV serum 2 of people | Without HBV DNA | 40.00 | Negative |
| Linear sensitivity reference material L1 | 7.6×10 7IU/ml | 24.45 | Positive |
| Linear sensitivity reference material L2 | 4.7×10 6IU/ml | 28.21 | Positive |
| Linear sensitivity reference material L3 | 6.7×10 5IU/ml | 31.06 | Positive |
| Linear sensitivity reference material L4 | 4.6×10 4IU/ml | 35.22 | Positive |
| Linear sensitivity reference material L5 | 4.3×10 3IU/ml | 37.22 | Positive |
| Negative quality control product | 3.2×10 6IU/ml | 28.79 | Positive |
| Positive quality control product 1 dilutes 10 2Doubly | 5.3×10 8IU/ml | 22.56 | Positive |
| Positive quality control product 2 dilutes 10 3Doubly | 2.5×10 7IU/ml | 25.78 | Positive |
| Positive quality control product 3 dilutes 10 4Doubly | 5.6×10 6IU/ml | 29.23 | Positive |
| Positive quality control product 4 dilutes 10 5Doubly | 1.4×10 5IU/ml | 32.24 | Positive |
| Positive quality control product 5 dilutes 10 6Doubly | 3.10×10 4IU/ml | 34.78 | Positive |
| Positive quality control product 6 dilutes 10 7Doubly | 5.1×10 3IU/ml | 37.52 | Positive |
| Positive quality control product 7 dilutes 10 8Doubly | 1.0-5.0×10 2IU/ml | 39.57 | Negative |
| Positive quality control product 8 dilutes 10 9Doubly | 1.0-5.0×10 2IU/ml | 39.57 | Negative |
In test kit, one group of detection reagent detected result feminine gender of hepatitis B virus core gene promoter DNA illustrates in the sample detected without hepatitis B virus DNA.The detected result positive illustrates in the sample detected to there is hepatitis B virus DNA.
Embodiment 2: one group of detection reagent of hepatitis B virus core gene promoter DNA in test kit, pcr amplification can be carried out smoothly to the A1762T saltant type of hepatitis B virus base, comprise following composition:
The negative quality control product of detection experiment: with embodiment 1.
The positive quality control product of detection experiment: with embodiment 1.
PCR reaction solution B:PCR damping fluid (1 ×), Taq DNA polymerase 1.5u, dNTP0.3 μm ol/L, and:
HBV-B1 upstream primer 5 '-tgggg gagga gatta ggtta at-3 ' 0.3 μm of ol/L
HBV-B2 upstream antisense primer 5 '-tttaa cctaa tctcc tcccc ca-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 '; 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 '; 0.2 μm of ol/L
Concrete implementation step:
1) sample DNA process: get negative quality control product and positive quality control product, use without the negative human serum Sample Dilution of hepatitis B virus to required concentration, get 30 μ l to add equivalent DNA extraction liquid 60 μ l and mix, 95-98 DEG C of sex change in 8--9 minute, place 0 DEG C, refrigerator cooling 2-3 minute, centrifugal 5 minutes of 12000rpm, gets supernatant liquor centrifugal for subsequent use.
2) PCR reaction solution packing: respectively get PCR reaction solution A and B, add each reaction tubes by the μ l packing of detection 1 person-portion 25 again, respectively get Detection and Extraction HBV DNA supernatant liquor 5 μ l to be measured and add each reaction tubes, every increment originally need detect with PCR reaction solution A and B two side reaction pipe simultaneously.
3) pcr amplification: get point each reaction tubes installing PCR reaction solution, increases by following pcr amplification condition setting: 94 DEG C of 3 minutes denaturations, then 93 DEG C of-55 DEG C of circulations in 60 seconds in 40 seconds 40 times.
Detected result and judgement:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
The positive quality control product of applying detection test, make different extent of dilution concentration, carry out minimal detectable concentration test, detected result, as following table, illustrates that minimal detectable concentration is 5.1 × 10
3iU/ml, CT are 35.46, and this susceptibility can meet hospital clinical inspection needs.
Result illustrates: positive in embodiment 1 detected result, under there is the precondition of hepatitis B virus DNA in judgement sample, although embodiment 2 detects negative findings and illustrates in the sample detected to there is hepatitis B virus DNA but A1762T without hepatitis B virus DNA suddenly change, detect positive findings and illustrate in the sample of detection have the A1762T of hepatitis B virus DNA to suddenly change.
Embodiment 3: one group of detection reagent of hepatitis B virus core gene promoter DNA in test kit, pcr amplification can be carried out smoothly to the Gl764A saltant type of hepatitis B virus base, comprise following composition:
The negative quality control product of detection experiment: with embodiment 1.
The positive quality control product of detection experiment: with embodiment 1.
PCR reaction liquid C: PCR damping fluid (1 ×), Taq DNA polymerase 1.5u, dNTP0.3 μm ol/L, and:
HBV-C1 upstream primer 5 '-ggagg agatt aggtt aaaga-3 ' 0.3 μm of ol/L
HBV-C2 upstream antisense primer 5 '-ccttt aacct aatct cctcc-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ', 0.2 μm of ol/L
Concrete implementation step:
1) sample DNA process: get negative quality control product and positive quality control product, use without the negative human serum Sample Dilution of hepatitis B virus to required concentration, get 30 μ l to add equivalent DNA extraction liquid 60 μ l and mix, 95-98 DEG C of sex change in 8--9 minute, place 0 DEG C, refrigerator cooling 2-3 minute, centrifugal 5 minutes of 12000rpm, gets supernatant liquor centrifugal for subsequent use.
2) PCR reaction solution packing: respectively get PCR reaction solution A and B, add each reaction tubes by the μ l packing of detection 1 person-portion 25 again, respectively get Detection and Extraction HBV DNA supernatant liquor 5 μ l to be measured and add each reaction tubes, every increment originally need detect with PCR reaction solution A and B two side reaction pipe simultaneously.
3) pcr amplification: get point each reaction tubes installing PCR reaction solution, increases by following pcr amplification condition setting: 94 DEG C of 3 minutes denaturations, then 93 DEG C of-55 DEG C of circulations in 60 seconds in 40 seconds 40 times.
Detected result and judgement:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
The positive quality control product of applying detection test, make different extent of dilution concentration, carry out minimal detectable concentration test, detected result, as following table, illustrates that minimal detectable concentration is 5.1 × 10
3iU/ml, CT are 35.67, and this susceptibility can meet hospital clinical inspection needs.
Result illustrates: positive in embodiment 1 detected result, under there is hepatitis B virus DNA precondition in judgement sample, although embodiment 3 detects negative findings and illustrates in the sample detected to there is hepatitis B virus DNA, but suddenly change without the G1764A of hepatitis B virus DNA, detect positive findings and illustrate in the sample detected have the G1762A of hepatitis B virus DNA to suddenly change.
Embodiment 4: A1762T, G1764A abrupt climatic change of the hepatitis B virus DNA of clinical sample
1) sample DNA process: get hepatitis B virus clinical serum sample 30 μ l and add equivalent DNA extraction liquid 60 μ l and mix, 95-98 DEG C of sex change in 8--9 minute, place 0 DEG C, refrigerator cooling 2-3 minute, centrifugal 5 minutes of 12000rpm, get supernatant liquor centrifugal for subsequent use, negative quality control product and the positive quality control product contrast of detection experiment are set simultaneously.
2) PCR reaction solution packing: respectively get PCR reaction solution A and B, add each reaction tubes by the μ l packing of detection 1 person-portion 25 again, respectively get Detection and Extraction HBV DNA supernatant liquor 5 μ l to be measured and add each reaction tubes, every increment originally need detect with PCR reaction solution A, B and C reaction tubes simultaneously.
3) pcr amplification: get point each reaction tubes installing PCR reaction solution, increases by following pcr amplification condition setting: 94 DEG C of 3 minutes denaturations, then 93 DEG C of-55 DEG C of circulations in 60 seconds in 40 seconds 40 times.
Detected result and judgement:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36.
DNA detection gray area: 36<CT value <38, needs recast once, as result >36 judges to detect feminine gender.
A1762T and the Gl764A saltant type result of Clinical Laboratory hepatitis B virus core gene promoter DNA hepatitis B virus base, as following table, illustrates, this specific performance meets hospital clinical inspection needs.
Pattern detection result illustrates:
The negative quality control product contrast of sequence 1. is wild type gene, and hepatitis B virus DNA detected result is positive, other two detected result feminine genders, occurs without sudden change.
The contrast of sequence 2. positive quality control product is mutated genes, and hepatitis B virus DNA detected result is positive, other two detected result positives, has two positions to suddenly change and occurs.
Sequence 3. clinical serum sample, hepatitis B virus DNA detected result total negative, without hepatitis B virus DNA, more occurs without sudden change.
Sequence 4. clinical serum sample, hepatitis B virus DNA detected result is positive, and other two detected result feminine genders, without hepatitis B virus DNA
Sudden change occurs.
Sequence 5. clinical serum sample, hepatitis B virus DNA detected result is positive, and A1762T detected result is positive, undergos mutation, and G1764A detected result is negative.
Sequence 6. clinical serum sample, hepatitis B virus DNA detected result is positive, and A1762T detected result is negative, G1764A detected result is positive, undergos mutation.
Sequence 7. clinical serum sample, hepatitis B virus DNA detected result is positive, and other two detected result positives, A1762T and G1764A undergos mutation simultaneously.
The pcr amplification product DNA of above clinical serum sample 4-7 is carried out its result of nucleotide sequencing as follows:
Clinical serum sample 4 nucleotide sequencing result:
gtttaaagac tgggaggagt tgggggagga gattaggtta aaggtctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct。
Sequencing results illustrates: this nucleotide sequence is without A1762T position and the variation of Gl764A bit base.
Clinical serum sample 5 nucleotide sequencing result:
gtttaaagac tgggaggagt tgggggagga gattaggtta atggtctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct。
Sequencing results illustrates: this nucleotides sequence shows the variation of A1762T bit base, and makes a variation without Gl764A bit base.
Clinical serum sample 6 nucleotide sequencing result:
gtttaaagac tgggaggagt tgggggagga gattaggtta aagatctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct。
Sequencing results illustrates: this nucleotide sequence without the variation of A1762T bit base, and has Gl764A bit base to make a variation.
Clinical serum sample 7 nucleotide sequencing result:
gtttaaagac tgggaggagt tgggggagga gattaggtta atgatctttg tactaggagg ctgtaggcat aaattggtct gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac tgttcaagcc tccaagctgt gccttgggtg gct。
Sequencing results illustrates: this nucleotide sequence has A1762T position and the variation of Gl764A bit base simultaneously.
Each clinical serum sample is through nucleotide sequencing interpretation of result, completely the same with this PCR kit detected result.
SEQUENCE LISTING
<110> Canvest (Wuhan) Biotechnology Co., Ltd.
<120> mono-kind detects test kit and the detection method of hepatitis B virus core gene promoter base mutation
<130> mono-kind detects test kit and the detection method of hepatitis B virus core gene promoter base mutation
<160> 7
<170> PatentIn version 3.1
<210> 1
<211> 20
<212> DNA
<213> synthetic
<400> 1
gtttaaagac tgggaggagt 20
<210> 2
<211> 22
<212> DNA
<213> synthetic
<400> 2
tgggggagga gattaggtta at 22
<210> 3
<211> 20
<212> DNA
<213> synthetic
<400> 3
tttaacctaa tctcctcccc 20
<210> 4
<211> 20
<212> DNA
<213> synthetic
<400> 4
ggaggagatt aggttaaaga 20
<210> 5
<211> 20
<212> DNA
<213> synthetic
<400> 5
cctttaacct aatctcctcc 20
<210> 6
<211> 21
<212> DNA
<213> synthetic
<400> 6
agccacccaa ggcacagctt g 21
<210> 7
<211> 22
<212> DNA
<213> synthetic
<400> 7
tgtaggcata aattggtctg tt 22
Claims (2)
1. detect a test kit for hepatitis B virus core gene promoter base mutation, it is characterized in that this test kit comprises following composition:
PCR kit A group reagent:
PCR reaction solution A:PCR damping fluid, Taq DNA polymerase, dNTP
HBV-A1 upstream primer 5 '-gttta aagac tggga ggagt-3 '
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 '
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ';
PCR kit B group reagent:
PCR reaction solution B:PCR damping fluid, Taq DNA polymerase, dNTP
HBV-B1 upstream primer 5 '-tgggg gagga gatta ggtta at-3 '
HBV-B2 upstream antisense primer 5 '-tttaa cctaa tctcc tcccc ca-3 '
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 '
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ';
PCR kit C group reagent:
PCR reaction liquid C: PCR damping fluid, Taq DNA polymerase, dNTP
HBV-C1 upstream primer 5 '-ggagg agatt aggtt aaaga-3 '
HBV-C2 upstream antisense primer 5 '-ccttt aacct aatct cctcc-3 '
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 '
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ';
Wherein:
PCR reaction solution A:1 × PCR damping fluid, Taq DNA polymerase 1.5U, dNTP 0.3 μm of ol/L;
HBV-A1 upstream primer 5 '-gttta aagac tggga ggagt-3 ' 0.3 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 '; 0.2 μm of ol/L;
PCR reaction solution B:1 × PCR damping fluid, Taq DNA polymerase 1.5U, dNTP 0.3 μm of ol/L
HBV-B1 upstream primer 5 '-tgggg gagga gatta ggtta at-3 ' 0.3 μm of ol/L
HBV-B2 upstream antisense primer 5 '-tttaa cctaa tctcc tcccc ca-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ', 0.2 μm of ol/L;
PCR reaction liquid C: 1 × PCR damping fluid, Taq DNA polymerase 1.5U, dNTP 0.3 μm of ol/L
HBV-C1 upstream primer 5 '-ggagg agatt aggtt aaaga-3 ' 0.3 μm of ol/L
HBV-C2 upstream antisense primer 5 '-ccttt aacct aatct cctcc-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ', 0.2 μm of ol/L.
2. a kind of test kit detecting hepatitis B virus core gene promoter base mutation according to claim 1, its detection method is:
1) sample DNA process: get test serum sample 30 μ l and add equivalent DNA extraction liquid 60 μ l and mix, 95-98 DEG C of sex change in 8-9 minute, places the cooling of 0 DEG C, refrigerator 2-3 minute, 12000rpm centrifugal 5 minutes, gets supernatant liquor centrifugal for subsequent use;
2) PCR reaction solution packing: respectively get each 25 μ l of PCR reaction solution A, B and C, gets Detection and Extraction HBV DNA supernatant liquor 5 μ l to be measured and adds each reaction tubes, and every increment originally need detect with PCR reaction solution A and B two side reaction pipe simultaneously;
PCR reaction solution A:1 × PCR damping fluid, Taq DNA polymerase 1.5U, dNTP 0.3 μm of ol/L;
HBV-A1 upstream primer 5 '-gttta aagac tggga ggagt-3 ' 0.3 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 '; 0.2 μm of ol/L;
PCR reaction solution B:1 × PCR damping fluid, Taq DNA polymerase 1.5U, dNTP 0.3 μm of ol/L
HBV-B1 upstream primer 5 '-tgggg gagga gatta ggtta at-3 ' 0.3 μm of ol/L
HBV-B2 upstream antisense primer 5 '-tttaa cctaa tctcc tcccc ca-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ', 0.2 μm of ol/L;
PCR reaction liquid C: 1 × PCR damping fluid, Taq DNA polymerase 1.5U, dNTP 0.3 μm of ol/L
HBV-C1 upstream primer 5 '-ggagg agatt aggtt aaaga-3 ' 0.3 μm of ol/L
HBV-C2 upstream antisense primer 5 '-ccttt aacct aatct cctcc-3 ' 0.03 μm of ol/L
The public downstream primer 5 ' of HBV-D-agcca cccaa ggcac agctt g-3 ' 0.3 μm of ol/L
HBV-Y fluorescent probe: 5 '-FAM-tgtag gcata aattg gtctg tt-TAMARA-3 ', 0.2 μm of ol/L;
3) pcr amplification: get point each reaction tubes installing PCR reaction solution, increases by following pcr amplification condition setting: 94 DEG C of 3 minutes denaturations, then 93 DEG C 40 seconds, 55 DEG C of circulations in 60 seconds 40 times;
4) detected result and judgement:
(1) judgement that PCR group reagent box A group reagent detects hepatitis B virus core gene promoter DNA is applied:
DNA detection is negative: CT value >=38;
DNA detection is positive: CT value <36;
DNA detection gray area: 36<CT value <38, needs recast once, and the result >36 that reforms judges to detect feminine gender;
(2) judgement that PCR kit B group reagent detects hepatitis B virus core gene promoter A1762T nucleotide variation is applied:
Abrupt climatic change is negative: PCR reaction solution B pipe detects CT value >=38
Abrupt climatic change is positive: PCR reaction solution B pipe detects CT value <36;
DNA detection gray area: 36<CT value <38, needs recast once, and the result >36 that reforms judges feminine gender;
(3) judgement that PCR kit C group reagent detects hepatitis B virus core gene promoter G1764A nucleotide variation is applied:
Abrupt climatic change is negative: PCR reaction liquid C pipe detects CT value >=38;
Abrupt climatic change is positive: PCR reaction liquid C pipe detects CT value <36;
DNA detection gray area: 36<CT value <38, needs recast once, and the detected result >36 that reforms judges feminine gender.
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| Title |
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| 反义抑制PCR检测乙肝病毒G1896A变异株方法的研究;黄治慧等;《数理医药学杂志》;20111231;第24卷(第3期);摘要、第296页1.2、第297页1.4、表1、第298页3 * |
| 黄庆等.HBV核心区基因突变的研究.《中华医院感染学杂志》.2009,第19卷(第14期),1793-1796. * |
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