CN106916905A - Invisible hepatitis B detection kit and its detection method - Google Patents
Invisible hepatitis B detection kit and its detection method Download PDFInfo
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- CN106916905A CN106916905A CN201710031599.9A CN201710031599A CN106916905A CN 106916905 A CN106916905 A CN 106916905A CN 201710031599 A CN201710031599 A CN 201710031599A CN 106916905 A CN106916905 A CN 106916905A
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Abstract
The present invention provides a kind of invisible hepatitis B detection kit and its detection method, and primer sets include sense primer CA, anti-sense primer CB, sense primer CC, anti-sense primer CD in the kit；The sequence of the sense primer CA such as SEQ ID NO：Shown in 1；The sequence of the anti-sense primer CB such as SEQ ID NO：Shown in 2；The sequence of the sense primer CC such as SEQ ID NO：Shown in 3；The sequence of the anti-sense primer CD such as SEQ ID NO：Shown in 4.The present invention redesigns the primer in Core/pre core areas by analyzing the nest-type PRC primer that existing invisible hepatitis B virus infection is diagnosed, and improves detection efficiency, so as to detect that invisible hepatitis B virus infection lays the foundation in population of China from now on.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of invisible hepatitis B detection kit and its detection
Method.
Background technology
Hepatitis type B virus (hepatitis B virus, HBV) infection has turned into serious global public health and has asked
Topic, the total crowd of the whole world once infection hepatitis B is up to 2,000,000,000, and annual to there are about the liver that 1,000,000 people are died from caused by hepatitis B hard
There is obvious areal variation in the diseases such as change, primary carcinoma of liver and hepatic failure, its prevalence.From 1992, China was by hepatitis B vaccine
Planned immunity for children is included, neonate's hepatitis B immune prevention work achieves important achievement.It is B-mode according to national population in 2006
Seroepidemiological study of viral hepatitis shows that crowd HBsAg carrying rates are 7.18%, and compared with 1992 9.75% have dropped
26.35%, wherein 1~4 years old crowd's hepatitis B surface antigen carrying rate is minimum, it is 0.96%.
Invisible hepatitis B virus infection (Occult Hepatitis B Virus Infection, OBI) refers to be held in liver
Renew in hepatitis B virus DNA, and HBV DNA in serum are in low-level (＜ 200IU/ml) or feminine gender, while can not detect
HBsAg.Invisible hepatitis B virus infection is susceptible to suffer from the Prevalent district crowd high of hepatitis B, invisible hepatitis B virus infection can also occur
In children after hepatitis B vaccination.
Since the seventies in last century, invisible hepatitis B virus infection is more and more paid attention to.Shahmoradi S etc.
People once report with nested PCR method detect Iran area HB vaccination children in invisible hepatitis B virus infection, altogether inspection
Measure 21 invisible hepatitis B virus infection children.Previous experiments are verified by the nested PCR method to document report, sent out
Existing Core/pre-core section amplifications are undesirable, therefore, existing invisible hepatitis B virus detecting method is not particularly suited for
Chinese population, poor sensitivity, it would be highly desirable to improve.
The content of the invention
The shortcoming of prior art, detects it is an object of the invention to provide a kind of invisible hepatitis B in view of the above
Kit and its detection method, the detection method for solving in the prior art to invisible hepatitis B are examined Chinese population
Disconnected result is undesirable, poor sensitivity the problems such as.
In order to achieve the above objects and other related objects, first aspect present invention is provided for the detection of invisible hepatitis B
Primer sets, including sense primer CA, anti-sense primer CB, sense primer CC, anti-sense primer CD；
The sequence of the sense primer CA such as SEQ ID NO：Shown in 1；
The sequence of the anti-sense primer CB such as SEQ ID NO：Shown in 2；
The sequence of the sense primer CC such as SEQ ID NO：Shown in 3；
The sequence of the anti-sense primer CD such as SEQ ID NO：Shown in 4.
Second aspect present invention provides a kind of kit for detecting invisible hepatitis B containing above-mentioned primer sets.
Further, the sense primer CA, anti-sense primer CB be first round specific primer, the sense primer CC,
Anti-sense primer CD is the second wheel specific primer.
Further, the kit also includes one or more combination in PCR premixed liquids, HBV DNA, water.
Preferably, to contain Taq archaeal dna polymerases, dNTPs, standard Taq enzyme reaction buffer, enzyme steady for the PCR premixed liquids
Determine one or more combination in agent.
It is highly preferred that the PCR premixed liquids are 2 × Taq PCR Master Mix.
Third aspect present invention provides the application of above-mentioned primer sets or kit in invisible hepatitis B is detected.
Further, at least comprise the following steps：
1) the HBV DNA in testing sample are extracted；
2) nest-type PRC reaction：Using the sense primer CA, anti-sense primer CB to step 1) obtain HBV DNA carry out
First round PCR is expanded, then with the amplified production of first round PCR as template, is carried out using the sense primer CC, anti-sense primer CD
Second wheel PCR amplifications；
3) Gel electrophoresis results according to pcr amplification product judge whether purpose band occur.
Further, step 1) described in testing sample be selected from serum, blood plasma, whole blood, pure viral cultures and carry this
The Vector factors of viroid.
Further, step 2) in, first round pcr amplification reaction condition is as follows：Predegeneration：Temperature is 94 DEG C, and the time is
3min；Denaturation：Temperature is 94 DEG C, and the time is 30s；Annealing：Temperature is 55 DEG C, time 30s；Extend：Temperature is 72 DEG C, and the time is
1min；By after the circulation that 34 denaturation annealing extend, re-extending, 72 DEG C of temperature, time 5min.
Further, step 2) in, the second wheel pcr amplification reaction condition is as follows：Predegeneration：Temperature is 94 DEG C, and the time is
3min；Denaturation：Temperature is 94 DEG C, and the time is 30s；Annealing：Temperature is 55 DEG C, time 30s；Extend：Temperature is 72 DEG C, and the time is
1min；By after the circulation that 34 denaturation annealing extend, re-extending, 72 DEG C of temperature, time 5min.
As described above, a kind of invisible hepatitis B detection kit of the invention and its detection method, have with following
Beneficial effect：The present invention redesigns Core/ by analyzing the nest-type PRC primer that existing invisible hepatitis B virus infection is diagnosed
The primer in pre-core areas, improves detection efficiency, so as to detect invisible hepatitis B virus infection in population of China from now on
Lay the foundation.
Brief description of the drawings
Fig. 1 is shown as embodiment of the present invention Core/pre-core areas primer C1, C3, C4 detection HBsAg feminine gender children's samples
Gel electrophoresis figure.
Fig. 2 a are shown as the gel electrophoresis figure of C1, C3, C4 primer detection of embodiment of the present invention P4.
Fig. 2 b are shown as the nucleotide sequence comparison figure of C1, C3, C4 primer detection of embodiment of the present invention P4.
The embodiment of the present invention that Fig. 3 is shown as newly designs Core/pre-core areas sense primers CA, anti-sense primer CB, upstream
Primer CC, anti-sense primer CD detect the gel electrophoresis figure of HBV positive samples.
Fig. 4 be shown as embodiment of the present invention Core/pre-core areas sense primers CA, anti-sense primer CB, sense primer CC,
The gel electrophoresis figure of anti-sense primer CD detection HBsAg feminine gender children's samples.
Fig. 5 a and Fig. 5 b are shown as 5 HBV Core/pre-core region nucleotide sequences of children in the embodiment of the present invention
Comparison chart.
Specific embodiment
Embodiments of the present invention are illustrated below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands other advantages of the invention and effect easily.The present invention can also be by specific realities different in addition
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
In comparison result shown in Fig. 2 b, Fig. 5 a and Fig. 5 b, strigula "-" represents blank, and dot " " is represented and HBV
C gene identical bases.
Main experiment reagent：Serum-virus DNA extraction kit, purchased from Qiagen companies；2×Taq PCR Master
Mix (article No. KT201-02, specification 5ml, contain dyestuff), 100bp marker, purchased from Tiangeng biochemical technology (Beijing) limited public affairs
Department；Agarose, purchased from HraGene companies；GoodViewTM nucleic acid dyes, purchased from Beijing SBS Genetech bio-engineering corporation.
Experimental specimen：The serum specimen 100 of the children of the HBsAg feminine genders that Children's Hospital Attached to Chongqing Medical Univ. is in hospital
Part, it includes condition has：(1) infant serum specimen HBsAg is negative；(2) its mother or/and father HBsAg are positive；(3) 3 are completed
Secondary hepatitis B vaccination；(4) without blood transfusion history；(5) without other hepatitis viruses such as HAV, HCV infection；(6) without HIV；(7) year
Age is more than August.Collect the serum specimen 1 of the positive children of HBV.
Serum extracts HBV DNA experiments：Using serum-virus DNA extraction kit, by kit operating instruction, blood is extracted
HBV DNA in clear sample.
Nested PCR experiments：The nested PCR amplification reaction system of 25 μ L is as follows：2×Taq PCR Master Mix 12.5μ
L, the μ L of forward primer 1, the μ L of reverse primer 1, DNA profiling (HBV DNA) 5 μ L, ddH2O 5.5μL。
Nest-type PRC response parameter is as follows：Predegeneration：Temperature is 94 DEG C, and the time is 3min；Denaturation：Temperature is 94 DEG C, time
It is 30s, annealing：Different reaction primer temperature is different, and the time is 30s, extends：Temperature is 72 DEG C, and the time is 1min, by 34
After the circulation that individual denaturation annealing extends；Re-extend, temperature is 72 DEG C, and the time is 5min.
The annealing temperature of different primers is different, and the annealing temperature in P areas and S areas is 62 DEG C, and the annealing temperature in X areas is 55
DEG C, the annealing temperature in C areas and PS areas is 55 DEG C of the first round, the second 62 DEG C of wheel；Primer sequence see the table below 1.
Table 1
Newly-designed Core/pre-core areas primer：The conserved region of the Core/pre-core areas gene of selection HBV DNA
Primer is designed in domain, is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Sense primer CA is located at the 1797-1815 base zones of Core/pre-core areas upstream, and sequence is：5’–
GTCTGTTCACCAGCACCAT-3’(SEQ ID NO：1)；
Anti-sense primer CB is located at the 2377-2395 base zones in Core/pre-core areas, and sequence is：5’–
TCTGCGAGGCGAGGGAGTT-3’(SEQ ID NO：2)；
Sense primer CC is located at the 1877-1896 base zones in Core/pre-core areas, and sequence is：5’–
CTGTGCCTTGGGTGGCTTTG-3’(SEQ ID NO：3)；
Anti-sense primer CD is located at the 2345-2362 base zones in Core/pre-core areas, and sequence is：5’–
CCTGCCTCGTCGTCTAAC-3’(SEQ ID NO：4).
First round pcr amplification reaction reagent is configured to：
First round pcr amplification reaction parameter is as follows：Predegeneration：Temperature is 94 DEG C, and the time is 3min；Denaturation：Temperature is 94
DEG C, the time is 30s, annealing：55 DEG C of first round annealing temperature, the time is 30s, is extended：Temperature is 72 DEG C, and the time is 1min, warp
After crossing the circulation that 34 denaturation annealing extend；Re-extend, temperature is 72 DEG C, and the time is 5min.
With first round PCR primer as template, the second wheel PCR reactions are carried out.
Second takes turns being configured to for pcr amplification reaction reagent：
| Composition | Final concentration | Mother liquid concentration | Consumption (μ L) |
| 2×Taq PCR Master Mix | 1× | 2× | 12.5 |
| Sense primer CC | 0.4μM | 10μM | 1 |
| Anti-sense primer CD | 0.4μM | 10μM | 1 |
| HBV DNA | — | — | 5 |
| — | — | 5.5 | |
| Cumulative volume | — | — | 25 |
Second wheel pcr amplification reaction parameter is as follows：Predegeneration：Temperature is 94 DEG C, and the time is 3min；Denaturation：Temperature is 94
DEG C, the time is 30s, annealing：Second 55 DEG C of annealing temperature of wheel, the time is 30s, is extended：Temperature is 72 DEG C, and the time is 1min, warp
After crossing the circulation that 34 denaturation annealing extend；Re-extend, temperature is 72 DEG C, and the time is 5min.
Two-wheeled nested PCR product enters row agarose gel electrophoresis experiment respectively, and GoodViewTM nucleic acid dye is added during glue
Material, agarose concentration is 1%, voltage 160V, electrophoresis time 20min, the observation experiment result in gel imaging system.It is expected real
Test result：There is band in 600bp in first round product, and the second wheel product band occurs in 500bp.
Experimental result is as follows：
1. the nest-type PRC result of original primer：With the HBV DNA in the primer detection HBV positive children serums of table 1, electricity
Swimming result be：Each area can amplify purpose band.
2. HBV DNA results in original primer detection HBsAg feminine gender children serum：With the primer detection HBsAg in table 1
HBV DNA in negative children serum, electrophoresis result：Core/pre-core areas expanding effect is bad, occur except purpose band with
Outer multi-ribbon is (see Fig. 1, note：1：ddH2O is compareed；2-11：10 HBsAg ' negative ' specimens；12：Negative control；13：HBsAg sun
Property children's sample；14：100bp marker).Only has P4 appearance in 100 Core/pre-core areas of children's sample weaker
Purpose band is (see Fig. 2 a, note：1：P4；2：Negative control；3：HBV positive samples；4：100bp marker), the gene sequence of sequencing
Row comparison result shows that P4 gene orders are shown in Fig. 2 b, and it is HBV gene sequence to compare discovery with the gene order of HBV C genotype
Row.1 sample is had in the i.e. 100 parts samples of HBsAg feminine genders amplify HBV C genes.
SEQ ID NO：5 show the HBV C gene orders for comparing；
SEQ ID NO：6 show the P4 gene orders for comparing, and the sequence is by original primer by nested PCR amplification
Sequencing afterwards is obtained.
4. the nest-type PRC result of Core/pre-core areas primer is newly designed：Sense primer CA, anti-sense primer CB are first
Wheel primer, sense primer CC, anti-sense primer CD are the second wheel primer, and two-wheeled nested PCR experiments are observed in gel imaging system
The electrophoresis result of HBV positive samples：There is band in 600bp in first round PCR primer, and the second wheel PCR primer bar occurs in 500bp
Band, i.e. the primer Successful amplification purpose band.Electrophoretogram is shown in Fig. 3, note：1：ddH2O is compareed；2：Negative control；3：The positive marks of HBV
This；4：100bp marker.
5. the HBV DNA results in Core/pre-core areas primer detection HBsAg feminine gender children serums are newly designed：With new
HBV DNA in design Core/pre-core areas primer detection HBsAg feminine gender children serums, the knot of contrast primer C1, C3, C4
Fruit finds that its electrophoresis result is：Only there is purpose band (see Fig. 4, note：1：ddH2O is compareed；2-11：10 negative marks of HBsAg
This；12：Negative control；13：HBsAg positive children's samples；14：100bp marker), there are 5 samples in 100 children's samples
There is purpose band, the gene order comparison result of sequencing shows sees Fig. 5 a and Fig. 5 b, the gene order ratio with HBV C genotype
Have 5 samples in the negative samples of part HBsAg of HBV gene sequence, i.e., 100 and amplify HBV C genes to finding that it is.
The sequencing result of 5 samples is as follows：
SEQ ID NO：7 show the P4 gene orders for comparing, and the sequence is passed through by the primer that the present embodiment is designed
Sequencing is obtained after nested PCR amplification；
SEQ ID NO：8 show the P17 gene orders for comparing, and the sequence is passed through by the primer that the present embodiment is designed
Sequencing is obtained after nested PCR amplification；
SEQ ID NO：9 show the P23 gene orders for comparing, and the sequence is passed through by the primer that the present embodiment is designed
Sequencing is obtained after nested PCR amplification；
SEQ ID NO：10 show the P29 gene orders for comparing, and the sequence is passed through by the primer that the present embodiment is designed
Cross after nested PCR amplification to be sequenced and obtain；
SEQ ID NO：11 show the P72 gene orders for comparing, and the sequence is passed through by the primer that the present embodiment is designed
Cross after nested PCR amplification to be sequenced and obtain.
Experimental result is analyzed as follows：
Invisible hepatitis B virus infection has been found to develop close phase with the generation of the disease such as cirrhosis, primary carcinoma of liver
Close, it is also possible to increase the risk that the patients such as blood transfusion, dialysis, liver transfer operation infect HBV, it has larger potential hazard.Concealment
Property hepatitis B virus infection is according to hepatitis B core antibody (Hepatits B core Antibody, HBcAb) and hepatitis B surface antibody
Whether (Hepatits B surface Antibody, HBsAb) is positive and can be divided into positive serology OBI and serology
Negative OBI, positive serology OBI are positive hepatitis B core antibody, companion or not positive with hepatitis B surface antibody；Serology Negative is
Both are all negative.
Detection HBV DNA are the goldstandards for detecting invisible hepatitis B virus infection at present.Invisible hepatitis B virus infection
Detection enters line sensitivity nest-type PRC high or real-time using the specific primer for the highly conserved region of HBV different genes
Quantitative PCR.General detection HBV gene at least 3 conservative regions of group, such as S areas, X areas, C areas in nest-type PRC detection.Because of nido
The sensitivity of PCR is higher, and in order to reduce the false positive phenomenon occurred in experimentation, experiment every time must set negative right simultaneously
According to and blank, when the result of negative control and blank is negative, the positive findings of sample can just be adopted.
Therefore, the present embodiment have collected 100 parts of HBsAg feminine gender children serum samples, by the HBV DNA that extract sample simultaneously
Nested PCR experiments are carried out, according to electrophoresis result and sequencing result, is found：When primer C1, C3, C4 amplification HBV C genes, only
There is purpose band in P4, and the purpose band is weaker, and sequencing obtains its gene order, by the gene sequence with HBV C genotype
Row are compared and found, the similitude of two sections of gene orders is higher, i.e., the gene order of P4 is HBV gene sequence.
Design of primers plays an important role during nested PCR experiments.PCR primer design is unreasonable, is easily caused experiment
There is deviation in result：Electrophoresis result show as it is amplifiable go out purpose band outside multiple bands, purpose band is weaker or does not go out
Existing purpose band etc..The present embodiment verified by primer C1, C3, the C4 to Core/pre-core areas, finds its electrophoresis knot
There is the multi-ribbon outside purpose band in fruit.Its primer is analyzed, it is found that the primer has problems with：(1) 3 ' ends of primer C1
There is the continuous bases G GG of more than 3, mistake may be caused to trigger probability increase；(2) the last bit base in 3 ' ends of primer C4 is A,
It causes amplification efficiency apparently higher than other bases in mismatch site；(3) G/C content of primer C3 is higher than 60%, and upstream and downstream is drawn
The G/C content of thing has big difference, and is unfavorable for the carrying out of PCR reactions.For problem above, while the HBV gene type point according to China
Cloth situation, the present embodiment redesigns the primer in Core/pre-core areas, and finds that the primer can be used to expand by verifying
HBV DNA。
The present embodiment detects 100 parts of HBsAg feminine gender children serum marks by with new design sense primer CA, CB, CC, CD
This, it is found that 5 sample Successful amplifications go out purpose band.Sequencing respectively obtain 5 gene orders of sample, by with HBV C bases
Because the gene order of type is compared, it is found that these gene orders are higher with the similitude of HBV, it is seen that point mutation, this 5 bases of sample
Because sequence is HBV gene sequence.It is newly-designed i.e. in the P4 samples that primer C1, C3, C4 Successful amplification goes out genes of interest
Also Successful amplification goes out genes of interest to CA, CB, CC, CD primer；Meanwhile, do not amplify 4 of genes of interest in primer C1, C3, C4
In sample, newly-designed CA, CB, CC, CD primer Successful amplification goes out genes of interest.Newly-designed CA, CB, CC, CD primer is significantly
Improve detection efficiency.
In sum, the present invention is analyzed according to the genotype of China HBV by the conserved sequence to HBV gene, from
And design the primer of the invisible hepatitis B virus infection diagnosis suitable for Chinese population.
The Core/pre-core region sequences of HBV 1 B genes type and C genotype are completely the same, therefore, primer master of the invention
To be applied to the diagnosis of HBV 1 B genes type and C genotype hepatitis B virus infections.
The above-described embodiments merely illustrate the principles and effects of the present invention, not for the limitation present invention.It is any ripe
The personage for knowing this technology all can carry out modifications and changes under without prejudice to spirit and scope of the invention to above-described embodiment.Cause
This, those of ordinary skill in the art is complete with institute under technological thought without departing from disclosed spirit such as
Into all equivalent modifications or change, should be covered by claim of the invention.
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http://www.nhfpc.gov.cn/jkj/s3582/201307/
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SEQUENCE LISTING
<110>Children's Hospital Attached to Chongqing Medical Univ.
<120>Invisible hepatitis B detection kit and its detection method
<130> PCQYK
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial
<220>
<223>Sense primer CA
<400> 1
gtctgttcac cagcaccat 19
<210> 2
<211> 19
<212> DNA
<213> Artificial
<220>
<223>Anti-sense primer CB
<400> 2
tctgcgaggc gagggagtt 19
<210> 3
<211> 20
<212> DNA
<213> Artificial
<220>
<223>Sense primer CC
<400> 3
ctgtgccttg ggtggctttg 20
<210> 4
<211> 18
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<213> Artificial
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cctgcctcgt cgtctaac 18
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<211> 639
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<213> Artificial
<220>
<223> HBV C
<400> 5
atgcaacttt ttcacctctg cctaatcatc tcatgttcat gtcctactgt tcaagcctcc 60
aagctgtgcc ttgggtggct ttggggcatg gacattgacc cgtataaaga atttggagca 120
tctgtggagt tactctcttt tttgccttct gacttctttc cttctattcg agatctcctc 180
gacaccgcct ctgctctgta tcgggaggcc ttagagtctc cggaacattg ttcacctcac 240
catacagcac tcaggcaagc tattctgtgt tggggtgagt taatgaatct ggccacctgg 300
gtgggaagta atttggaaga cccagcatcc agggaattag tagtcagcta tgtcaatgtt 360
aatatgggcc taaaaatcag acaactattg tggtttcaca tttcctgtct tacttttgga 420
agagaaactg ttcttgagta tttggtgtct tttggagtgt ggattcgcac tcctcccgct 480
tacagaccac caaatgcccc tatcttatca acacttccgg aaactactgt tgttagacga 540
cgaggcaggt cccctagaag aagaactccc tcgcctcgca gacgaaggtc tcaatcgccg 600
cgtcgcagaa gatctcaatc tcgggaatct caatgttag 639
<210> 6
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ctcataatgt gccttgggtg gctttggggc atggacattg acccgtataa agaatttgga 60
gcttctgtgg agttactctc ttttttgcct tctgacttct ttccttctat tcgagatctc 120
ctcgacaccg cctctgctct gtatcgggag gccttagagt ctccggaaca ttgttcacct 180
caccatacgg cactcaggca agctatcctg tgttggggtg agttgatgaa tctagccacc 240
tgggtgggaa gtaatttgga agatccagca tccagggaat tagtagtcag ctatgtcaac 300
gttaatatgg gcctaaaaat cagacaacta ttgtggtttc acatttcctg tcttactttt 360
gggagagaaa ctgttcttga atatttggtg tcttttggag tgtggattcg cactcctcct 420
gcatatagac caccaaatgc ccctatctta tcaacacttc 460
<210> 7
<211> 432
<212> DNA
<213> Artificial
<220>
<223> P4CD
<400> 7
agataggggc atttggtggt ctatatgcag gaggagtgcg aatccacact ccaaaagaca 60
ccaaatattc aagaacagtt tctctcccaa aagtaagaca ggaaatgtga aaccacaata 120
gttgtctgat ttttaggccc atattaacgt tgacatagct gactactaat tccctggatg 180
ctggatcttc caaattactt cccacccagg tggctagatt catcaactca ccccaacaca 240
ggatagcttg cctgagtgcc gtatggtgag gtgaacaatg ttccggagac tctaaggcct 300
cccgatacag agcagaggcg gtgtcgagga gatctcgaat agaaggaaag aagtcagaag 360
gcaaaaaaga gagtaactcc acagaagctc caaattcttt atacgggtca atgtccatgc 420
cccaaagcca ca 432
<210> 8
<211> 437
<212> DNA
<213> Artificial
<220>
<223> P17CD
<400> 8
gataagatag gggcatttgg tggtctgtaa gcaggaggag tgcgaatcca cactccaaaa 60
gataccaaat actcaaggac agtttctctt ccaaaagtaa gacaggaaat gtgaaaccac 120
agtagttgtc tgatttttag gcccatgtta acattgacat agctgactac taattccctg 180
gatgctgggt cttccaaatt acttcccacc caggtggcca gattcatcaa ctcaccccaa 240
cacagaatag cttgcctgag tgcagtatgg tgaggtgagc aatgttccgg agactctaag 300
gcctcccgat atagagcaga ggcggtgtcg aggagatctc gaatagaagg aaagaagtca 360
gacggcaaaa aagagagtaa ctccacagaa gctccaaatt ctttatacgg gtcaatgtcc 420
atgccccaaa gccacaa 437
<210> 9
<211> 434
<212> DNA
<213> Artificial
<220>
<223> P23CD
<400> 9
tgatagatag gggcatttgg tggtctgtaa gcaggaggag tgcgaatcca cactccaaaa 60
gataccaaat actcaaggac agtttctctt ccaaaagtaa gacaggaaat gtgaaaccac 120
agtagttgtc tgatttttag gcccatgtta acattgacat agctgactac taattccctg 180
gatgctgggt cttccaaatt acttcccacc caggtggcca gattcatcaa ctcaccccaa 240
cacagaatag cttgcctgag tgcagtatgg tgaggtgagc aatgttccgg agactctaag 300
gcctcccgat atagagcaga ggcggtgtcg aggagatctc gaatagaagg aaagaagtca 360
gacggcaaaa aagagagtaa ctccacagaa gctccaaatt ctttatacgg gtcaatgtcc 420
atgccccaaa gccc 434
<210> 10
<211> 430
<212> DNA
<213> Artificial
<220>
<223> P29CD
<400> 10
agataggggc atttggtggt ctgtaagcag gaggagtgcg aatccacacc ccaaaagata 60
ccaaatactc aagggcagtt tctcttccaa aagtaagaca ggaaatgtga aaccacagta 120
gttgtctgat ttttaggccc atgttaacat tgacatagct gactactaat tccctggatg 180
ctgggtcttc caaattactt cccacccagg tggccagatt catcaactca ccccaacaca 240
gaatagcttg cctgagtgca gtatggtgag gtgagcaatg ttccggagac tctaaggcct 300
cccgatatag agcagaggcg gtgtcgagga gatctcgaat agaaggaaag aagtcagacg 360
gcaaaaaaga gagtaactcc acagaagctc caaattcttt atacgggtca atgtccatgc 420
cccaaagccc 430
<210> 11
<211> 426
<212> DNA
<213> Artificial
<220>
<223> P72CD
<400> 11
ggggcatttg gtggtctgta agcaggagga gtgcgaatcc acactccaaa agataccaaa 60
tactcaagga cagtttctct tccaaaagta agacaggaaa tgtgaaacca cagtagttgt 120
ctgattttta ggcccatgtt aacattgaca tagctgacta ctaattccct ggatgctggg 180
tcttccaaat tacttcccac ccaggtggcc agattcatca actcacccca acacagaata 240
gcttgcctga gtgcagtatg gtgaggtgag caatgttccg gagactctaa ggcctcccga 300
tatagagcag aggcggtgtc gaggagatct cgaatagaag gaaagaagtc agacggcaaa 360
aaagagagta actccacaga agctccaaat tctttatacg ggtcaatgtc catgccccaa 420
agcccc 426
Claims (10)
1. it is a kind of for invisible hepatitis B detection primer sets, it is characterised in that including sense primer CA, anti-sense primer
CB, sense primer CC, anti-sense primer CD；
The sequence of the sense primer CA such as SEQ ID NO：Shown in 1；
The sequence of the anti-sense primer CB such as SEQ ID NO：Shown in 2；
The sequence of the sense primer CC such as SEQ ID NO：Shown in 3；
The sequence of the anti-sense primer CD such as SEQ ID NO：Shown in 4.
2. the kit for detecting invisible hepatitis B of primer sets according to claim 1 is contained.
3. kit according to claim 2, it is characterised in that：The sense primer CA, anti-sense primer CB are the first round
Specific primer, the sense primer CC, anti-sense primer CD are the second wheel specific primer.
4. kit according to claim 2, it is characterised in that：The kit also include PCR premixed liquids, HBV DNA,
One or more combination in water.
5. kit according to claim 4, it is characterised in that：The PCR premixed liquids contain Taq archaeal dna polymerases,
One or more combination in dNTPs, standard Taq enzyme reaction buffer, enzyme stabilizers, it is preferable that the PCR premixed liquids are 2
×Taq PCR Master Mix。
6. primer sets according to claim 1 or the kit described in claim any one of 2-5 are detecting invisible second
Application in hepatovirus.
7. application according to claim 6, it is characterised in that：At least comprise the following steps：
1) the HBV DNA in testing sample are extracted；
2) nest-type PRC reaction：Using the sense primer CA, anti-sense primer CB to step 1) obtain HBV DNA carry out first
Wheel PCR amplifications, then with the amplified production of first round PCR as template, second is carried out using the sense primer CC, anti-sense primer CD
Wheel PCR amplifications；
3) Gel electrophoresis results according to pcr amplification product judge whether purpose band occur.
8. application according to claim 7, it is characterised in that：Step 1) described in testing sample be selected from serum, blood plasma, complete
Blood, pure viral cultures and carry the Vector factors of this viroid.
9. application according to claim 7, it is characterised in that：Step 2) in, first round pcr amplification reaction condition is as follows：
Predegeneration：94 DEG C of temperature, time 3min；Denaturation：94 DEG C of temperature, time 30s；Annealing：55 DEG C of temperature, time 30s；Extend：Temperature
72 DEG C of degree, time 1min；By after the circulation that 34 denaturation annealing extend, re-extending, 72 DEG C of temperature, time 5min.
10. application according to claim 7, it is characterised in that：Step 2) in, the second wheel pcr amplification reaction condition is as follows：
Predegeneration：94 DEG C of temperature, time 3min；Denaturation：94 DEG C of temperature, time 30s；Annealing：55 DEG C of temperature, time 30s；Extend：Temperature
72 DEG C of degree, time 1min；By after the circulation that 34 denaturation annealing extend, re-extending, 72 DEG C of temperature, time 5min.
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| CN201710031599.9A CN106916905B (en) | 2017-01-17 | 2017-01-17 | Invisible hepatitis B detection kit and its detection method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710031599.9A CN106916905B (en) | 2017-01-17 | 2017-01-17 | Invisible hepatitis B detection kit and its detection method |
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| CN106916905B CN106916905B (en) | 2018-06-08 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109852732A (en) * | 2019-04-02 | 2019-06-07 | 重庆医科大学附属儿童医院 | The invisible hepatitis B detection kit of quantitative fluorescent PCR |
| CN113549707A (en) * | 2020-04-24 | 2021-10-26 | 利多(香港)有限公司 | Method, oligonucleotide and kit for HBV genotype detection |
| CN117431338A (en) * | 2023-10-25 | 2024-01-23 | 深圳市血液中心(深圳市输血医学研究所) | Kit and method for detecting hepatitis B virus based on nested PCR and fluorescent PCR |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852732A (en) * | 2019-04-02 | 2019-06-07 | 重庆医科大学附属儿童医院 | The invisible hepatitis B detection kit of quantitative fluorescent PCR |
| CN113549707A (en) * | 2020-04-24 | 2021-10-26 | 利多(香港)有限公司 | Method, oligonucleotide and kit for HBV genotype detection |
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| EP4141129A4 (en) * | 2020-04-24 | 2024-06-05 | Leadway (HK) Limited | Method for detecting hbv genotype, oligonucleotide and kit |
| CN117431338A (en) * | 2023-10-25 | 2024-01-23 | 深圳市血液中心(深圳市输血医学研究所) | Kit and method for detecting hepatitis B virus based on nested PCR and fluorescent PCR |
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| Publication number | Publication date |
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| CN106916905B (en) | 2018-06-08 |
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