CN104388598B - A kind of HBV cccDNA digital pcr immue quantitative detection reagent box and application thereof - Google Patents
A kind of HBV cccDNA digital pcr immue quantitative detection reagent box and application thereof Download PDFInfo
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Abstract
The invention belongs to the outer nucleic acid detection technique field of virion, be specially a kind of HBV cccDNA digital pcr immue quantitative detection reagent box and application thereof.First the present invention designs HBV cccDNA digital pcr detection by quantitative probe compositions, and test kit includes the container containing detection probe compositions and containing liver puncture tissue DNA extract purified reagent container.The step that this test kit uses includes: extracts DNA in liver puncture tissue, and uses PSAD enzyme to digest HBV rcDNA；With digital pcr detection chip as reaction vessel, composite fluorescence multiple PCR primer is used in digital pcr chip detection micropore, target DNA to be expanded；According to the fluorescence signal in each micropore of data PCR detection chip, obtain the quantitative result of HBV cccDNA in each cell.High specificity of the present invention, highly sensitive, easy, quick, it is not necessary to HBV cccDNA and HBV rcDNA is carried out extraction, overcome the defect in tradition HBV cccDNA detection, be suitable to large-scale promotion application.
Description
Technical field
The invention belongs to the outer nucleic acid detection technique field of virion, be specifically related to HBV cccDNA digital pcr detection by quantitative
Test kit and application thereof.
Background technology
Chronic hepatitis B (Chronic Hepatitis B, CHB) is due to hepatitis B virus (Hepatitis B
Virus, HBV) a kind of inflammation disease caused by chronic infection in human liver cell, its continuing advances may result in liver cirrhosis,
Liver failure and hepatocarcinoma.According to estimates, the whole world has HBV carrier about 3.5 hundred million people at present, there are about 1,000,000 people every year and dies from HBV
Liver failure, liver cirrhosis or hepatocarcinoma caused by infection.2012, hepatopathy branch of Chinese Medical Association and Chinese Medical Association infect disease and learn
Branch combines " the guideline " of formulation and points out, the existing Patients with Chronic HBV Infection of current China about 93,000,000,
Wherein CHB patient is more than 20,000,000.
For CHB, " the guideline " that either the still Europe hepatopathy association of China works out refers both to
Going out, antiviral therapy is most important remedy measures.In the antiviral therapy of CHB, nucleoside (sour) analog (includes rummy husband
Fixed, adefovir ester, Sebivo, Entecavir, for Nuo Fuwei ester etc.) definitely become with its convenient oral, Anti-viral Treatment
The class medicine being most widely used in CHB patient, wherein Entecavir and tenofovir disoproxil are the one of domestic and international guide recommendation
Line medication.
Either using nucleoside (sour) analog or interferon etc. to carry out Anti-HBV activity treatment, Anti-viral Treatment monitoring is all adopted
Detect the virus load in CHB peripheral blood in patients with traditional real time fluorescence quantifying PCR method, i.e. HBV DNA is quantitative
Detection.When using nucleoside (sour) analog to carry out Anti-HBV activity treatment, associated treatment guide both domestic and external is all pointed out, need to be by CHB
HBV DNA in peripheral blood in patients controls the level that can't detect in existing detection means.But, the equal table of research both domestic and external
Bright, people's liver cell is only main host cell and the main place of hbv replication of HBV infection, in human peripheral
HBV come from hbv replication in hepatocyte row outer with secretion.In human liver cell, the starting template of hbv replication is not
Lax ring-type HBV DNA(i.e. HBV rcDNA in the ripe HBV granule of the outer row of secretion), but a kind of virus covalently closed circular HBV
DNA(i.e. HBV cccDNA).In other words, the only HBV cccDNA in hepatocyte thoroughly removes, and just can be shown that chronic type b liver
Inflammation is cured.In human peripheral, HBV DNA quantitatively curative effect monitoring index as antiviral therapy is reliable.But, when
Use tradition real-time PCR detection peripheral blood HBV DNA for HBV cccDNA in Patients ' Hepatocytes not being described time negative
Existence.Therefore during long-term antiviral therapy, especially when using traditional real-time fluorescence PCR technology for detection to carry
Showing when peripheral blood HBV DNA is negative, in peripheral blood, the monitoring of HBV DNA is whole for judgement, the treatment of medicine late result
The selection meaning of point is limited.This is also to control for nucleoside (sour) analog Anti-HBV activity in current associated treatment guide the most at home and abroad
One of clear and definite major reason for the treatment of terminal is not all given during treatment.Currently, for CHB patient, use nucleoside (sour) analog
Anti-HBV activity treatment time without the clear and definite course for the treatment of, i.e. cannot drug withdrawal, this become educational circles common recognition.
The major reason causing this situation is in Anti-HBV activity therapeutic process, when peripheral blood HBV DNA is negative
After, there is no effective means at present and the amount of the hbv replication template HBV cccDNA in hepatocyte is carried out accurate evaluation.The most clinical
On have use In situPCR method to detect the distribution scenario of HBV DNA in liver puncture tissue.But, this method operates
Complicated, sensitivity is low and cannot distinguish between HBV rcDNA and the HBV cccDNA in hepatocyte.
The difficult point of HBV cccDNA detection is how to evade " non-specific amplification " of HBV rcDNA to HBV cccDNA
The impact of testing result.The sequence of HBV cccDNA and HBV rcDNA is consistent, therefore, and " the non-spy of so-called HBV rcDNA
Specific amplification " primer non-specific amplification the most truly.Research finds, HBV cccDNA and both HBV rcDNA
Structurally there is significant difference.HBV rcDNA minus strand is relatively complete, only at HBV DNA direct repeat sequence 1(Direct
Repeats 1, DR1) one little breach of upstream, district existence, normally referred to as " nick ", i.e. incise；Its normal chain then exists
One big breach, normally referred to as " gap ".The most there is not above-mentioned incising or breach in HBV cccDNA, positive and negative chain is all in completely
Ring-type, it is similar to plasmid.The HBV cccDNA detection method of most scholar's designs is i.e. based on HBV rcDNA and HBV
Difference in this structure of cccDNA, by HBV design of primers in the gap regions of HBV rcDNA normal chain and the downstream incised, inspection
Probing pin designs in the downstream of primer.On the basis of this design, then it is aided with mung-bean nuclease or PSAD enzyme etc. and can degrade
The nuclease digestion HBV rcDNA of single stranded DNA, thus HBV cccDNA is purified process with the phase reaching HBV cccDNA
To specific detection.But, although so, there are some researches show, at total HBV quantitative result 1 × 105More than copies/ml
Time, still cannot avoid the impact that HBV cccDNA is detected by HBV rcDNA, because, in traditional real-time PCR detection system
In, HBV cccDNA and HBV rcDNA is simultaneous.And substantial amounts of research shows, in hepatocyte, the amount of HBV rcDNA is led to
Often it is about more than 1000 times of HBV cccDNA.
Currently, for CHB antiviral therapy, when drug withdrawal can become a most key problem, also be current
One hot issue of educational circles's research both at home and abroad.In view of China, CHB patient base is huge and a line Anti-HBV activity such as Entecavir
Medicine is expensive, and according to life-long therapy scheme, the most individual or social undertaken heath economics burden is all
Inestimable.And this problem is carried out effectively, the research of science, HBV cccDNA is special, detection by quantitative instrument accurately
To be indispensable means.Therefore, HBV cccDNA in hepatocyte all can be carried out determining by domestic and international educational circles in the urgent need to one
Quick, easy, special, the sensitive method of amount.
Summary of the invention
Present invention aims to present on current demand and tradition based on real-time fluorescence PCR quantitative technique
Deficiency etc. method detection HBV cccDNA, it is provided that a kind of HBV cccDNA digital pcr immue quantitative detection reagent box and application thereof.
First the present invention designs HBV cccDNA digital pcr detection by quantitative probe compositions, including: a PCR reaction is drawn
, the 2nd PCR reaction primer pair and the 3rd PCR react primer pair by thing, and corresponding detection probe and competitiveness probe.Its
In:
Described oneth PCR reaction primer to (i.e. detection HBV cccDNA specific primer to) be SEQ ID No. 1
(i.e. P3+P9+P1) and the SEQ ID i.e. P4+P2 of No. 2() shown in nucleotide sequence；The corresponding spy detecting HBV cccDNA
Specific probes is the SEQ ID i.e. P5 of No. 5() shown in nucleotide sequence；
2nd PCR reaction primer to (i.e. detection reference gene RPP40 primer to) be the SEQ ID i.e. P3+P6 of No. 6()
With the SEQ ID i.e. P4+P7 of No. 7() shown in nucleotide sequence；The specific probe of corresponding detection reference gene RPP40 is
The SEQ ID i.e. P8 of No. 8() shown in nucleotide sequence；
(i.e. in HBV cccDNA and reference gene RPP40 amplified reaction common primers to) is by the 3rd PCR reaction primer
The SEQ ID i.e. P3 of No. 3() and the SEQ ID i.e. P4 of No. 4() shown in nucleotide sequence；
Described competitive probe is the SEQ ID i.e. P10 of No. 10() shown in nucleotide sequence.
Wherein, wherein P1 section and P2 section are HBV specific sequence；P6 section and P7 section are that people single copy gene RPP40 is special
Property sequence.
Nucleotide sequence (i.e. P9) shown in SEQ ID No.9, for the link sequence between P3, P1.
SEQ ID No.1 to SEQ ID No.10 nucleotide sequence and decorative features are shown in Table 1.
Table 1 SEQ ID No.1 to SEQ ID No.10 nucleotide sequence and modification
| SEQ ID | Nucleotide sequence (5`-3`) and decorative features |
| SEQ ID No. 1 | CCATCTCATCCCTGCGTGTCTCGTAGACTAGACTCATCTGCCGGACCGTGT |
| SEQ ID No. 2 | CCTCTCTATGGGCAGTCGGTGATCTTGGAGGCTTGAACAGTAGGACAT |
| SEQ ID No. 3 | CCATCTCATCCCTGCGTGTCTC |
| SEQ ID No. 4 | CCTCTCTATGGGCAGTCGGTGAT |
| SEQ ID No. 5 | FAM-TAGACTAGACTCATCTGCCG-MGB |
| SEQ ID No. 6 | CCATCTCATCCCTGCGTGTCTCTGGCTGTGAACAAGGCTGGAT |
| SEQ ID No. 7 | CCTCTCTATGGGCAGTCGGTGATCAACAGCCCTAGCATCGTGATGT |
| SEQ ID No. 8 | VIC-AGGAAGGGTAGGGAGAC-MGB |
| SEQ ID No. 9 | GTAGACTAGA |
| SEQ ID No. 10 | GCAGAGGTGAAGCGAAGTCdd |
Remarks: Cdd is double dideoxycytosine.
In the present invention, for improving the specificity of HBV cccDNA detection, design competing based on the DR2 sequence of HBV DNA
Striving property probe P10(SEQ ID No. 10), its 3 ' end is through terminating modification.This sequence can with the DR2 in HBV DNA and on
Trip complementary combines, but does not extends.
In the present invention, HBV cccDNA detects probe P5(SEQ ID No. 5) to be positioned at HBV in SEQ ID No. 1 special
Property binding fragment P1 before rather than downstream, the composite sequence that this probe is HBV specific fragment P1 to be formed with random fragment P9,
And before this probe is positioned at HBV specific binding fragment P1 amplification starting point.Only in the presence of HBV cccDNA,
Forming a PCR and react complete amplicon, when there is the complementary strand of nucleotide sequence shown in SEQ ID No.1, P5 just can draw
Detection signal is discharged under the effect of thing SEQ ID No.3 (P3).Non-exponential amplified production to the HBV rcDNA of high concentration
P5 does not produce detection signal.
HBV cccDNA digital pcr immue quantitative detection reagent box of the present invention, including: primer and probe container, liver is worn
Thorn tissue DNA extract purified reagent container.Above-mentioned HBV cccDNA digital pcr is contained respectively quantitative in primer and probe container
Detection probe compositions, particularly, described primer and probe container are contained within SEQ ID No. 1, SEQ ID No. 2, SEQ
ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and
The solution of SEQ ID No.10；PSAD enzyme, PSAD enzyme buffer liquid and ATP is included in described DNA extraction thing purified reagent container
Solution.
In described test kit, SEQ ID No.1, SEQ ID No.2, SEQ ID No.6, SEQ ID No.7, SEQ ID
No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.8 working concentration scope are followed successively by: 35～65nmol/L, 35～
65nmol/L, 35～65nmol/L, 35～65nmol/L, 350～650nmol/L, 350～650nmol/L, 100～
175nmol/L, 100～175nmol/L, the reaction density of competitive probe SEQ ID No.10 is 200～400nmol/L；If
Meter concentration is 10 times of reaction densities.
The invention still further relates to above-mentioned HBV cccDNA digital pcr immue quantitative detection reagent box, at chronic hepatitis B patient
Application in HBV cccDNA detection by quantitative.Specifically comprise the following steps that
(1) extract DNA in liver puncture tissue, and use PSAD enzyme that HBV rcDNA is digested；
(2) with digital pcr detection chip as reaction vessel, above-mentioned HBV cccDNA digital pcr quantitative detecting reagent is used
Box, uses composite fluorescence multiple PCR primer to expand target DNA in digital pcr chip detection micropore:
The primer of the oneth PCR reaction is to for P3+P9+P1 total length (SEQ ID No. 1), P4+P2 total length (SEQ ID No.
2)；
The primer of the 2nd PCR reaction is to for P3+P6 total length (SEQ ID No. 6), P4+P7 total length (SEQ ID No. 7)；
The primer of the 3rd PCR reaction quotes P3(SEQ ID No. 3 to for public) and P4(SEQ ID No. 4)；
With the product of a PCR reaction as template, P3, P4 and fluorescein FAM(blue-fluorescence) the probe P5(SEQ of labelling
ID No. 5) produce HBV cccDNA specific detectable signal, with the 2nd PCR reaction product as template, P3, P4 and fluorescein
VIC(green fluorescence) the probe P8(SEQ ID No. 8 of labelling) produce RPP40 specific detectable signal.Detection due to chip
Number cells is far above HBV cccDNA molecular number in reaction system, and the detection signal in each micropore is the list of corresponding template and divides
Son detection signal；
(3) according to the fluorescence signal in each micropore of data PCR detection chip, blue-fluorescence number cells is overall reaction body
HBV cccDNA quantitative result in system, the green fluorescence number cells of 0.5 times is in overall reaction system detected cell number, blue
Color fluorescence number cells and the ratio of 0.5 times of green fluorescence micropore are in liver puncture specimen HBV cccDNA in average each cell
Quantitative result.RPP40 gene test signal is simultaneously as the inner quality control of PCR amplification system.
Extract DNA in liver puncture tissue described in step of the present invention (1), be to use QIAamp DNA mini Kit examination
Agent box extracts STb gene from hepatic tissue, including HBV cccDNA, HBV rcDNA and human gene group DNA；Extract is used
PSAD enzymic digestion HBV rcDNA, system is as follows:
DNA extraction thing 32 μ l
PSAD enzyme (10U/ μ l) 2.0 μ l
10 × enzyme cutting buffering liquid 4.0 μ l
ATP(25mM) 2.0 μl
Cumulative volume is 40 microlitres, 37 DEG C of incubations 60 minutes, then 95 DEG C of incubations 10 minutes.
The invention has the beneficial effects as follows:
(1) in the present invention, after HBV cccDNA detection probe is placed in common primers, HBV specific binding fragment
Before amplification starting point, thus the non-HBV cccDNA signal that the non-exponential amplification avoiding high concentration HBV rcDNA produces；
(2) have employed digital pcr and unconventional real-time PCR detection system, due to digital pcr detection chip micropore
Number is much larger than HBV cccDNA copy number, it is ensured that each the HBV that the micropore corresponding of blue-fluorescence copies occurs
CccDNA, detection by quantitative will be converted to the qualitative detection in each detection micropore；
(3) it is the specificity improving HBV cccDNA detection further, reaction system has added what 3 ' ends were modified through termination
Competitive probe P10(SEQ ID No. 10), this sequence DR2 district and upstream sequence thereof can be combined but not prolong in HBV DNA
Stretch, thus avoid the self annealing of HBV rcDNA non-exponential amplified fragments；
(4) detection sensitivity is high, owing to digital pcr detection chip number cells is much larger than HBV cccDNA copy number, permissible
Ensure that each the HBV cccDNA of the corresponding copy of the micropore of blue-fluorescence occurs.Both made detection system only has a copy
HBV cccDNA also can detect.
Detection design of the present invention is ingenious, high specificity, highly sensitive, detects quick, easy, and being suitable to large-scale promotion should
With.
Accompanying drawing explanation
Fig. 1: HBV cccDNA detection primer design drawing.Accompanying drawing show HBV rcDNA structural representation, above straight line show
HBV rcDNA normal chain, direction is from 5 ' ends to 3 ' ends, and dotted portion is shown as " breach " big in HBV rcDNA normal chain；The most straight
The minus strand of line display HBV rcDNA and little " incising " thereon.Nucleotides sequence shown in SEQ ID No.1 is classified as P3+P9+P1,
Wherein P1 section is the one section of HBV specific fragment being combined with HBV minus strand, is positioned at HBV rcDNA normal chain and surpasses the upstream at top
In " breach ", P5 is detection probe, and the part held including P1 section 5 ' and a P9 section 3 ' end part, P9 is a random sequence.
For HBV rcDNA, owing to P5 is positioned at P1 upstream, even if there is high concentration HBV rcDNA, the non-exponential that P1 is caused expands also
Do not produce detection signal.All disappear only for breach shown in dotted line in HBV cccDNA(now figure and incising of minus strand), SEQ
Primer shown in ID No. 2 (i.e. P4+P2) will form a complete amplicon, this amplicon with primer shown in SEQ ID No. 1
The complementary strand complete by comprising nucleotide sequence shown in SEQ ID No. 1, under P3, P4 primer amplification, probe P5 discharges inspection
Survey signal designation HBV cccDNA.The competitive probe designed based on DR2 region shown in P10, can be with the DR2 of HBV DNA
Region and upstream sequence thereof combine, but do not extend.
Detailed description of the invention
Content for a better understanding of the present invention, is described further below in conjunction with specific embodiment.Should be understood that following tool
Body embodiment is merely illustrative the present invention rather than limitation of the present invention.
Embodiment 1: the preparation of DNA in liver puncture tissue
Liver organization puncture is carried out by medical treatment routine.Take 5mm and puncture the liver organization obtained, shred with eye scissors, 200 μ
L PBS washs, and 800 revs/min centrifugal removes supernatant, totally 3 times.Use QIAGEN genome little liver organization after washing
Amount extraction agent box extracting genomic DNA, eluent 96 μ L.
Extract uses PSAD enzymic digestion HBV rcDNA, and system is as follows:
DNA extraction thing 32 μ l
PSAD enzyme (10U/ μ l) 2.0 μ l
10 × enzyme cutting buffering liquid 4.0 μ l
ATP(25mM) 2.0 μl
Cumulative volume is 40 microlitres, 37 DEG C of incubations 60 minutes, then 95 DEG C of incubations 10 minutes.
The design of embodiment 2:HBV cccDNA and RPP40 specific primer probe and synthesis
DR2 region upstream, the HBV rcDNA normal chain gap regions of HBV DNA, guarding according to different genotype HBV
Property, design HBV specific primer fragment P1, at its 5 ' end one section of random sequence P9(SEQ ID No. 9 of link), in P9 upstream
Link public forward primer P3(SEQ ID No.3) form nucleotide sequence shown in SEQ ID No. 1, this nucleotides sequence afterwards
Row are HBV cccDNA specific upstream primer.5 ' 11 nucleotide sequences of end of P1 are formed with 3 ' 9 nucleotide of end of P9
HBV cccDNA specificity detection probe P5(SEQ ID No.5), its 5 ' end flag F AM fluorescein, 3 ' end labelling MGB.At HBV
RcDNA minus strand incises upstream, according to the conservative of different genotype HBV, designs HBV specific primer fragment P2, at its 5 ' end
Link public forward primer P4(SEQ ID No. 4) form nucleotide sequence shown in SEQ ID No. 2, this nucleotides sequence afterwards
Row are HBV cccDNA specific Down Stream primer.Specific primer fragment is designed in human genome RPP40 gene conserved regions
Nucleotides sequence shown in the SEQ ID No. 6 formed after P6 and P7, P6 link P3 is classified as RPP40 forward primer, shape after P7 link P4
Nucleotides sequence shown in the SEQ ID No. 7 become is classified as RPP40 downstream primer, in RPP40 forward primer downstream according to sequence preservative
Property design RPP40 specific probe P8(SEQ ID NO. 8) and be MGB at its 5` end mark fluorescent element VIC, 3` end.For entering one
Step improves specificity, the ID No. of design competition probe P10(SEQ based on the DR2 region of HBV DNA 10).SEQ ID
No. 1 to SEQ ID No. 10 nucleotide sequence and decorative features are shown in Table 2.
Table 2:SEQ ID No. 1 to SEQ ID No. 10 nucleotide sequence and decorative features
Embodiment 3:HBV cccDNA detection by quantitative
(1) PCR reaction system:
By preparation digital pcr reaction system shown in table 3；
Table 3 HBV cccDNA digital pcr detection by quantitative reaction system
(2) detection chip loading:
By digital pcr user's manual requirement, the PCR reactant liquor prepared is loaded onto reaction core by chip loading hole
Sheet, uses 5.50 μ L confining liquid capping chips subsequently；
(3) PCR reaction condition:
Reaction condition as shown in table 4 carries out PCR amplification；
Table 4 HBV cccDNA digital pcr detection by quantitative reaction condition
(4) deciphering of digital pcr result:
In reaction system total HBV cccDNA quantitatively=blue-fluorescence number cells；
In reaction system total cell number quantitatively=0.5 × green fluorescence number cells；
95% credibility interval of above-mentioned quantitative result uses Possion distribution to estimate.
HBV cccDNA copy number=blue-fluorescence number cells/(0.5 × green fluorescence number cells) in average every cell.
The present invention is organized as detecting sample with chronic hepatitis B patient liver puncture, and sample, after PBS rinses, uses
QIAGEN genome extraction agent box in a small amount is to the DNA in detection sample, and extract, after PSAD ferment treatment, uses composite fluorescence
HBV cccDNA and human genome single copy gene RPP40 is examined in digital pcr detection chip micropore by multiplex PCR parallel
Survey.Owing to the number cells in digital pcr detection chip is total much larger than the HBV cccDNA copy number in total detection system and cell
Number, the detection signal in each micropore is single copy target molecule detection signal.Can be real by counting positive signal number cells
Now to HBV cccDNA and the counting of cell.Owing to people's cell is diploid cell, then the green number cells of 0.5 times is system
Middle detected total cellular score.When blue number cells is more than 10 000, re-start detection after template DNA should be diluted 100 times.
The test kit that the present invention provides can complete at the laboratory with digital pcr, and detection needs to use digital pcr
Detection chip completes, and the detection time only needs 3 to 5 hours；Result interpretation is intuitive and reliable；This invention can have with steady implementation
The technical conditions of standby large-scale promotion.
Therefore, the test kit that the present invention provides can produce in biotech company and detect in biomedicine easily
Mechanism is used for detecting, and possesses Industry Promotion condition.
In sum, the HBV cccDNA digital pcr immue quantitative detection reagent box design of the present invention is ingenious, high specificity, spirit
Sensitivity is high, and detects quick, accurate, easy, can be used for chronic viral hepatitis B and suffers from the quantitative inspection of HBV cccDNA in liver puncture tissue
Survey, be suitable to large-scale promotion application.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still may be made that
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, description is considered to be illustrative rather than limiting
Property.
<110>Shanghai Wuseshi Medical Research Co., Ltd.
<120>a kind of HBV ccc DNA digital pcr immue quantitative detection reagent box and application thereof
<160> 10
<210> 1
<211> 51
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(51)
<223>HBV cccDNA detects forward primer
<400> 1
CCATCTCATC CCTGCGTGTC TCGTAGACTA GACTCATCTG CCGGACCGTG T 51
<210> 2
<211> 48
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(48)
<223>HBV cccDNA detects downstream primer
<400> 2
CCTCTCTATG GGCAGTCGGT GATCTTGGAG GCTTGAACAG TAGGACAT 48
<210> 3
<211> 22
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(22)
<223>public forward primer
<400> 3
CCATCTCATC CCTGCGTGTC TC 22
<210> 4
<211> 23
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(23)
<223>public downstream primer
<400> 4
CCTCTCTATG GGCAGTCGGT GAT 23
<210> 5
<211> 20
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(20)
<223>HBV cccDNA detects probe, 5 ' end mark fluorescent element FAM, 3 ' end labelling MGB
<400> 5
TAGACTAGAC TCATCTGCCG 20
<210> 6
<211> 43
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(43)
<223>people RPP40 detects forward primer
<400> 6
CCATCTCATC CCTGCGTGTC TCTGGCTGTG AACAAGGCTG GAT 43
<210> 7
<211> 46
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(46)
<223>people RPP40 detects downstream primer
<400> 7
CCTCTCTATG GGCAGTCGGT GATCAACAGC CCTAGCATCG TGATGT 46
<210> 8
<211> 17
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(17)
<223>people RPP40 detects probe, 5` end mark fluorescent element VIC, 3` end labelling MGB
<400> 8
AGGAAGGGTA GGGAGAC 17
<210> 9
<211> 10
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(10)
<223>linking between public forward primer P3 and HBV specific fragment P1 uses random sequence
<400> 9
GTAGACTAGA 10
<210> 10
<211> 19
<212> DNA
<213>artificial sequence
<220>
<221> misc_feature
<222> (1)...(19)
<223>competitive probe, 3 ' ends are double dideoxycytosine
<400> 10
GCAGAGGTGA AGCGAAGTC 19
Claims (4)
1. a HBV cccDNA digital pcr immue quantitative detection reagent box, it is characterised in that including: primer and probe container, liver
Puncturing tissue DNA extraction thing purified reagent container；Described primer and probe container are contained within SEQ ID No. 1, SEQ ID No.
2、SEQ ID No. 3、SEQ ID No. 4、SEQ ID No. 5、SEQ ID No. 6、SEQ ID No. 7、SEQ ID No.
The solution of 8 and SEQ ID No.10；Include in described DNA extraction thing purified reagent container PSAD enzyme, PSAD enzyme buffer liquid and
ATP solution；Wherein:
SEQ ID No. 1 i.e. P3+P9+P1 and SEQ ID No. 2 i.e. nucleotides sequence shown in P4+P2 is classified as a PCR reaction
The primer specific primer pair to i.e. detecting HBV cccDNA；SEQ ID No. 5 i.e. nucleotides sequence shown in P5 is classified as corresponding inspection
Survey the specific probe of HBV cccDNA；
SEQ ID No. 6 i.e. P3+P6 and SEQ ID No. 7 i.e. nucleotides sequence shown in P4+P7 is classified as the 2nd PCR and reacts primer
To the primer pair i.e. detecting reference gene RPP40；SEQ ID No. 8 i.e. nucleotides sequence shown in P8 is classified as and detects internal reference accordingly
The specific probe of gene RPP40；
SEQ ID No. 3 i.e. P3 and SEQ ID No. 4 i.e. nucleotides sequence shown in P4 is classified as the 3rd PCR reaction primer to i.e.
Common primers pair in HBV cccDNA and reference gene RPP40 amplified reaction；
SEQ ID No. 10 i.e. nucleotides sequence shown in P10 is classified as HBV competitiveness probe.
HBV cccDNA digital pcr immue quantitative detection reagent box the most according to claim 1, it is characterised in that SEQ ID
No.1、SEQ ID No.2、SEQ ID No.6、SEQ ID No.7、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、
SEQ ID No.8 working concentration scope is followed successively by: 35～65nmol/L, 35～65nmol/L, 35～65nmol/L, 35～
65nmol/L, 350～650nmol/L, 350～650nmol/L, 100～175nmol/L, 100～175nmol/L, competitive spy
The reaction density of pin SEQ ID No.10 is 200～400nmol/L.
3. HBV cccDNA digital pcr immue quantitative detection reagent box as claimed in claim 1 or 2 is preparing chronic hepatitis B trouble
Application in person's HBV cccDNA immue quantitative detection reagent box, it is characterised in that adopt and detect with the following method:
(1) extract DNA in liver puncture tissue, and use PSAD enzyme that HBV rcDNA is digested；
(2) with digital pcr detection chip as reaction vessel, use HBV cccDNA digital pcr immue quantitative detection reagent box, use multiple
Close Multiplex fluorescent PCR primer in digital pcr chip detection micropore, target DNA to be expanded:
The primer of the oneth PCR reaction is to for SEQ ID No. 1, SEQ ID No. 2；
The primer of the 2nd PCR reaction is to for SEQ ID No. 6, SEQ ID No. 7；
The primer of the 3rd PCR reaction is to for the public SEQ ID No. 3 i.e. P4 of i.e. P3 and SEQ ID No. 4 quoted；
With the product of a PCR reaction as template, P3, P4 and fluorescein FAM label probe P5 i.e. SEQ ID No. 5 produce HBV
CccDNA specific detectable signal, with the product of the 2nd PCR reaction as template, P3, P4 and fluorescein VIC label probe P8 are i.e.
SEQ ID No. 8 produces RPP40 specific detectable signal；Owing to the detection number cells of chip is far above HBV in reaction system
CccDNA molecular number, the detection signal in each micropore is the Single Molecule Detection signal of corresponding template；
(3) according to the fluorescence signal in each micropore of data PCR detection chip, blue-fluorescence number cells is in overall reaction system
HBV cccDNA quantitative result, the green fluorescence number cells of 0.5 times is in overall reaction system detected cell number, blue glimmering
Light number cells and the ratio of 0.5 times of green fluorescence micropore are in liver puncture specimen determining of HBV cccDNA in average each cell
Amount result；RPP40 gene test signal is simultaneously as the inner quality control of PCR amplification system.
Application the most according to claim 3, it is characterised in that extract DNA in liver puncture tissue described in step (1), be
Use QIAamp DNA mini Kit test kit to extract STb gene from hepatic tissue, including HBV cccDNA, HBV rcDNA and
Human gene group DNA；Extract is used PSAD enzymic digestion HBV rcDNA, and system is as follows:
DNA extraction thing 32 μ l
PSAD enzyme, 10U/ μ l 2.0 μ l
10 × enzyme cutting buffering liquid 4.0 μ l
ATP, 25mM 2.0 μ l
Cumulative volume is 40 microlitres, 37 DEG C of incubations 60 minutes, then 95 DEG C of incubations 10 minutes.
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| CN105441595B (en) * | 2016-01-28 | 2018-11-16 | 安徽理工大学 | It is a kind of for detecting the digital pcr absolute quantitation parting detecting reagent of HBV-B/C |
| CN107541569A (en) * | 2016-06-24 | 2018-01-05 | 上海市公共卫生临床中心 | HBV DNA and cccDNA hybridization in situ detection kit in a kind of hepatic tissue |
| CN106381345A (en) * | 2016-12-07 | 2017-02-08 | 重庆医科大学附属第二医院 | Digital PCR (Polymerase Chain Reaction) detection probe, primer pair and detection method for hepatitis B virus |
| CN106636466A (en) * | 2016-12-30 | 2017-05-10 | 南方医科大学南方医院 | Precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA) |
| CN108441578A (en) * | 2017-02-15 | 2018-08-24 | 复旦大学附属华山医院 | A kind of digital droplet PCR detection method and its kit of hepatitis B virus cccDNA |
| CN107435083A (en) * | 2017-09-04 | 2017-12-05 | 上海市第十人民医院 | A kind of reliable clinical HBV DNA samples quantitative detecting method |
| CN108285931B (en) * | 2018-03-30 | 2021-01-01 | 武汉大学 | Micro-drop digital PCR method and kit for clinical detection of HBV cccDNA |
| JP2022546443A (en) | 2019-08-27 | 2022-11-04 | エフ．ホフマン－ラ ロシュ アーゲー | Compositions and methods for amplification and detection of hepatitis B virus RNA, including HBV RNA transcribed from cccDNA |
| CN110592289A (en) * | 2019-10-31 | 2019-12-20 | 苏州健雄职业技术学院 | High-sensitivity HBV DNA digital PCR quantitative detection kit and application thereof |
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| CN102329888A (en) * | 2010-11-05 | 2012-01-25 | 上海复星医学科技发展有限公司 | Fluorescent quantitative PCR (Polymerase Chain Reaction) testing method for cccDNA (covalently closed circular deoxyribonucleic acid) of hepatitis B virus and kit thereof |
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