CN106916908A - The enrichment extracting method and detection primer group, probe and method of HBV cccDNA - Google Patents
The enrichment extracting method and detection primer group, probe and method of HBV cccDNA Download PDFInfo
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Abstract
The invention belongs to biological technical field, and in particular to field of virus detection, primer sets, probe and the method for a kind of enrichment extracting method of HBV cccDNA and detection HBV cccDNA are more particularly to.Detection primer group is primer sets 1 or primer sets 2.Primer sets 1 are made up of following 4 primer sequences：Primer 1：Its nucleotide sequence such as SEQ ID NO：1；Primer 2：Its nucleotide sequence such as SEQ ID NO：2；Primer 3：Its nucleotide sequence such as SEQ ID NO：3；Primer 4：Its nucleotide sequence such as SEQ ID NO：4.Primer sets 2 are made up of following 4 primer sequences：Primer 5：Its nucleotide sequence such as SEQ ID NO：Shown in 5；Primer 6：Its nucleotide sequence such as SEQ ID NO：Shown in 6；Primer 7：Its nucleotide sequence such as SEQ ID NO：Shown in 7；Primer 8：Its nucleotide sequence such as SEQ ID NO：Shown in 8.Compared to the prior art, the method that the present invention is provided has accuracy higher, specificity and sensitivity.
Description
Technical field
The invention belongs to biological technical field, and in particular to field of virus detection, a kind of HBV cccDNA are more particularly to
Enrichment extracting method and the primer sets of detection HBV cccDNA, probe and method.
Background technology
Hepatitis type B virus (Hepatitis B virus, HBV) infection is in worldwide prevalence, and its infected is more than two
1000000000, it is that China endangers one of infectious disease of most serious at present.National hepatitis B epidemiology survey in 2006 shows that China is current
Still there are the people of hepatitis B surface antigen carrier about 93,000,000, wherein chronic hepatitis B patient about 20,000,000, hepatitis B and liver cancer patient
Account for a quarter in the whole world.Chronic HBV infection is cause chronic hepatitis, cirrhosis, hepatic failure and primary carcinoma of liver main
Reason, and cause more than 100 ten thousand people dead every year, cause great social danger.
HBV is hepadnavirus, is the pathogen for causing virus B hepatitis (hereinafter referred to as " hepatitis B ").It is extracellular
Hepatitis B virus DNA is a kind of double-stranded DNA of lax ring-type (relaxed circular DNA, rcDNA) molecule, its two
Chain is not closure, and wherein minus strand is more long, about 3200 bases, the full-length gene containing Hepatitis B virus-DNA, its 5 '
Have between initiating terminal and 3 ' ends one " incising " (nick) of several bases；Normal chain is shorter, there is larger " breach " (gap),
Its 3 ' end is not fixed, therefore length is variable, about the 50%~100% of minus strand length.In the reproduction process of hepatitis B
In, viral DNA enters host cell nuclear, and in the presence of archaeal dna polymerase, two breach of chain form supercoil by polishing
It is covalent, closure, ring-shaped DNA molecule (covalently closed circular DNA, cccDNA), before hepatitis B
The primary template that geneome RNA is replicated, starts a series of reproduction process.Although its content is less, in each liver cell only
About 5~50 copies, but duplication to hepatitis B and the foundation of Infection Status tool be of great significance.Hbv replication
It is a kind of conservative mechanism, it is still complete in transcription rear pattern plate cccDNA, and can transcribe repeatedly, so cccDNA is thermophilic hepatopathy
The key factor of malicious persistent infection.CccDNA long half times, it is impossible to thoroughly removed from vivo, there is many by long-term anti-in clinic
, after stopping antiviral therapy, and often there is HBV reactivations and disease relapse, main cause in the patient of viral therapy complete response
The lasting presence of cccDNA and it is difficult to clean off.It is to evaluate HBV infection state and medicine therefore cccDNA is the rising mark of hbv replication
The most important index of thing curative effect.Endonuclear cccDNA is only removed, hepatitis B patient virus could be thoroughly eliminated and be carried shape
State, is the target of antiviral therapy.
The eighties in last century, there is scholar to pass through DNA electrophoresis and Southern hybridization techniques, it was demonstrated that the presence of cccDNA
(Miller R H,Robinson W S.Hepatitis B virus DNA forms in nuclear and
cytoplasmic fractions of infected human liver[J].Virology,1984,137(2):390-
, but the method is cumbersome, sensitivity is low, poor accuracy 399.).The nineties in last century, round pcr starts to be applied to
The detection of cccDNA, establishes selective PCR detection technique, and introduction of competition PCR on this basis, realize it is preliminary it is quantitative (J,Schlicht H J.Analysis of the earliest steps of hepadnavirus
replication:genome repair after infectious entry into hepatocytes does not
depend on viral polymerase activity[J].Journal of virology,1993,67(8):4867-
4874.；Addison W R,Wong W W S,Fischer K P,et al.A quantitative competitive PCR
assay for the covalently closed circular form of the duck hepatitis B virus
[J].Antiviral research,2000,48(1):27-37.), to the beginning of this century, real-time fluorescence quantitative PCR inspection is established
Survey method (He M L, Wu J, Chen Y, the et al.A new and sensitive method for the of cccDNA
quantification of HBV cccDNA by real-time PCR[J].Biochemical and biophysical
research communications,2002,295(5):1102-1107.), quantifying truly is realized.Meanwhile,
Also there is other technologies method to be applied to the report of the detection of cccDNA, such as invade analytic approach, chimeric primers method, rolling circle amplification skill
(Jun-Bin S, Zhi C, Wei-Qin N, the et al.A such as art (rolling circle amplication, RCA)
quantitative method to detect HBV cccDNA by chimeric primer and real-time
polymerase chain reaction[J].Journal of virological methods,2003,112(1):45-
52.；Wong D K H,Yuen M F,Yuan H J,et al.Quantitation of covalently closed
circular hepatitis B virus DNA in chronic hepatitis B patients[J].Hepatology,
2004,40(3):727-737.；Zhong Y,Hu S,Xu C,et al.A novel method for detection of
HBV cccDNA in hepatocytes using rolling circle amplification combined with in
situ PCR[J].BMC infectious diseases,2014,14(1):1.；Margeridon S,Carrouée-
Durantel S,Chemin I,et al.Rolling circle amplification,a powerful tool for
genetic and functional studies of complete hepatitis B virus genomes from
low-level infections and for directly probing covalently closed circular DNA
[J].Antimicrobial agents and chemotherapy,2008,52(9):3068-3073.) existing cccDNA inspections
The main problems faced of survey technology is specificity and sensitivity, and this also causes the correlative study result of cccDNA to there is very big striving
View.For example, using identical or different technology, for the inspection of cccDNA in Peripheral Blood in Patients with Chronic Hepatitis B monocyte or serum
Survey has completely different report.To detection technique query in itself, very big puzzlement is caused to the correlative study of cccDNA,
Also the research of HBV medicines is further hindered.The maximum difficult point for setting up cccDNA detection techniques be in vivo have largely with
The HBV DNA of cccDNA sequence homologies other forms higher are present, including：①rcDNA；2. 5%~10% virus
Double-stranded linear DNA molecule present in particle (double-strand linear DNA, dsl-DNA)；3. by pregenome RNA
Reverse transcription forms DNA (single-stranded DNA, ssDNA).But in the cell in the HBV DNA of numerous forms,
CccDNA only accounts for a minimum part for total amount.
Selective fluorescent quantitative PCR technique has high sensitivity, and the principle of its detection cccDNA is based on rcDNA
There is breach in normal chain and minus strand, therefore can design across two primers of breach, rcDNA is not amplified, and
CccDNA is due to being that complete duplex structure then can be expanded selectively.For dsl-DNA or ssDNA, due to two primers
It is in opposite direction, will not also be amplified.But, when rcDNA templates amount is higher in reaction system, it is with incomplete two chains
Template formed single stranded extension products between can self-annealing (self-annealing) and cause false positive results (Zhang Y
Y,Zhang B H,Theele D,et al.Single-cell analysis of covalently closed circular
DNA copy numbers in a hepadnavirus-infected liver[J].Proceedings of the
National Academy of Sciences,2003,100(21):12372-12377).Other, such as chimeric primers method and invade
The Main Basiss for entering analytic approach differentiation rcDNA and cccDNA are that the normal chain of rcDNA is imperfect, thus chimeric primers or initial probe
Complementary therewith can not combine.But, two methods of dsl-DNA molecules can be what is be amplified.RCA can only expand ring-type
DNA profiling, it turned out that it can effectively distinguish cccDNA and rcDNA, compares many scholars and is detected with this method in recent years
CccDNA, but this method time-consuming (at least more than 4h) and it is quantitative to realize.
To overcome the defect of above-mentioned technical method, the mode being most frequently with document report is by taking effective measures
The content of other forms HBV DNA is reduced, so as to eliminate their influences to cccDNA detections of cccDNA.It has been reported that working as HBV
DNA is 107Copy/ml (containing) below when, can prevent selective fluorescence quantitative PCR detection from false positive occur.Have scholar according to
The characteristic distributions of cccDNA, cccDNA is only located in nucleus, and other several DNA moleculars are respectively positioned in cytoplasm, using thin
Karyon isolation technics, nucleus is separated carries out cccDNA detections (Song Peixin, Li Jun hepatitis B virus cccDNAs inspection
Survey method and clinical application research progress foreign medical science epidemiology pestology fascicle 2005；32:350-355.).Separately
Outward, cccDNA can not be with protein binding, and other three kinds of DNA are respectively positioned in nucleocapsid protein, and end is covalently tied with P albumen
Close, it is easy to combined with lauryl sodium sulfate (SDS), plus precipitation is formed after potassium chloride, (Kock can be separated by centrifugation
J,Theilmann L,Galle P,et al.Hepatitis B virus nucleic acids associated with
human peripheral blood mononuclear cells do not originate from replicating
virus.Hepatology,1996；23:405-13.).Further, cccDNA can not be by some enzymes such as mung-bean nuclease (mung
Bean nuclease, MBN), the ATP of non-degradable plasmid rely on DNA enzymatic (Plasmid-Safe ATP-dependent DNase,
The degraded such as PSAD), and the DNA of other forms is degradable.In above-mentioned processing method, ferment treatment is one kind side the most simple and effective
Formula, PSAD ferment treatments are using a kind of most enzymes.But increasing digestion step in cccDNA detections can make sample dilutions, together
When endonuclease reaction liquid in composition be likely to suppression PCR reaction, false negative result can be increased, be unfavorable for the detection of trace sample.
The content of the invention
In order to solve the above problems, the invention provides a kind of enrichment extracting method of HBV cccDNA and for detecting
The primer sets of HBV cccDNA, probe, and a kind of method for detecting HBV cccDNA is constructed, the method has high sensitive
Property, the sample of low concentration can be detected, lowest detection is limited to 60 copies/mL.
Term：" V/V " in the present invention refers to volume-volumetric concentration unit；" W/V " refers to quality-volumetric concentration.
On one side, the invention provides a kind of detection primer group and detection probe for detecting HBV cccDNA.
Described detection primer group is primer sets 1 or primer sets 2.
Described primer sets 1 are made up of the complementary series of following 4 primer sequences or following 4 primer sequences：
Primer 1：Its nucleotide sequence such as SEQ ID NO：Shown in 1,5 '-ACT CCC CGT CTG TGC CTT CT-3 '；
Primer 2：Its nucleotide sequence such as SEQ ID NO：Shown in 2,5 '-TTC TTT ATA CGG GTC AAT GTC CA-3 '；Primer
3：Its nucleotide sequence such as SEQ ID NO：Shown in 3,5 '-AGG AGG CTG TAG GCA TAA AT-3 '；Primer 4：Its nucleosides
Acid sequence such as SEQ ID NO：Shown in 4,5 '-TCC CGA TAC AAA GCA GAG-3 '.
Described primer sets 2 are made up of the complementary series of following 4 primer sequences or following 4 primer sequences：Primer 5：
Its nucleotide sequence such as SEQ ID NO：Shown in 5,5 '-ACG ACC GAC CTT GAG GCA TAC TTC-3 '；Primer 6：Its
Nucleotide sequence such as SEQ ID NO：Shown in 6,5 '-ATT CAT CAA CTC ACC CCA ACA CAG-3 '；Primer 7：Its core
Nucleotide sequence such as SEQ ID NO：Shown in 7,5 '-TAG GAG GCT GTA GGC ATA AAT-3 '；Primer 8：Its nucleotides sequence
Row such as SEQ ID NO：Shown in 8,5 '-CAA AGC AGA GGC GGT GTC-3 '.
The nucleotides sequence of described detection probe is classified as SEQ ID NO：9 (5 '-CTGTTCAAGCCTCCAAGCTG-3 ') institutes
The nucleotide sequence for showing or its complementary series；5 ' ends of the nucleotide sequence of described detection probe are marked with fluorophor, institute
The fluorophor stated is the one kind in FAM, HEX, VIC, CY5 and TET；What 3 ' ends of described detection probe were marked with is quenched base
Group, described quenching group is the one kind in TAMRA, MGB and BHQ.
Above-mentioned detection primer group and detection probe are to be designed to complete according to HBV cccDNA sequence characteristics, be can be used in real time
Quantitative fluorescent PCR (QPCR) technology, realizes the detection to HBV cccDNA.
On the other hand, the invention provides a kind of kit for detecting HBV cccDNA, described kit bag
Include above-mentioned detection primer group and detection probe.
In a preferred embodiment, described kit also includes internal control primer sets and internal control probe, described
Internal control primer sets and internal control probe as positive reference, for the quality control to kit,
Described internal control primer sets include lower two primers：
Primer 9：Its nucleotide sequence such as SEQ ID NO：Shown in 10,5 '-CGG GGT CAC CCA CAC TGT GCC
CAT CTA CG-3’；Primer 10：Its nucleotide sequence such as SEQ ID NO：Shown in 11,5 '-GGT CAC CCA CAC TGT
GCC CAT CTA CG-3’；
The sequence of described internal control probe is SEQ ID NO：12(5’-ATG CCC TCC CCC ATG CCA TCC T-
3 ') nucleotide sequence or its complementary series shown in；5 ' ends of the nucleotide sequence of described internal control probe are marked with fluorescent base
Group, described fluorophor is the one kind in FAM, HEX, VIC, CY5 and TET；What 3 ' ends of described internal control probe were marked with
Quenching group, described quenching group is the one kind in TAMRA, MGB and BHQ, and the fluorophor on described internal control probe should
Different from the fluorophor in detection probe.
Another aspect, the invention provides a kind of enrichment extracting method of HBV cccDNA, described enrichment extraction side
Method is comprised the following steps：
(1) sample to be tested is obtained：After test serum sample is shredded, it is put into centrifuge tube, is cracked to being added in centrifuge tube
Liquid, Proteinase K and magnetic bead, are vortexed and 50~60 DEG C of oscillation incubations are placed in after mixing, and incubation is completely dissolved to hepatic tissue, that is, is treated
Test sample sheet；If serum sample：Serum sample is then taken to be put into centrifuge tube, in centrifuge tube add lysate, Proteinase K and
Magnetic bead, is vortexed after mixing and is placed in 50~60 DEG C of oscillation incubations 15 minutes, that is, obtain sample to be tested；
(2) sample to be tested that step (1) is obtained is transferred on magnetic frame to place and collects within 5-10 minutes magnetic bead, centrifugation is abandoned in suction
Supernatant in pipe；
(3) collection obtained to step (2) adds eluent 1 in having the centrifuge tube of magnetic bead, is vortexed after mixing, by centrifuge tube
It is transferred on magnetic frame to place and collects within 3-5 minutes magnetic bead, the supernatant that suction is abandoned in centrifuge tube；
(4) collection obtained to step (3) adds eluent 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifuge tube is turned
To move to placed on magnetic frame and collect within 3-5 minutes magnetic bead, the supernatant that suction is abandoned in centrifuge tube；
(5) collection obtained to step (4) adds eluent 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifuge tube is turned
To move to placed on magnetic frame and collect within 3-5 minutes magnetic bead, the supernatant that suction is abandoned in centrifuge tube；
(6) collection obtained to step (5) adds digestion treatment fluid in having the centrifuge tube of magnetic bead, is vortexed after mixing, 37 DEG C
After being incubated 1h, 30min is incubated in 70 DEG C immediately；
(7) after the drop on the of short duration centrifugation tube wall being collected by centrifugation after step (6) is incubated, centrifuge tube is transferred to magnetic force
Placed 1 minute on frame, all of raffinate is abandoned in suction, open centrifuge tube lid and dry 5-10 minutes；
(8) eluent 3 is added in the dried centrifuge tube for obtaining to step (7), is vortexed after mixing, stand 3-10 points
Clock, centrifuge tube is transferred on magnetic frame to place 3-5 minutes and collects magnetic bead, separates supernatant；
(9) supernatant that step (8) is obtained is transferred in new centrifuge tube, is stored in -20 DEG C or is directly used in sample-adding.
Lysate in described enrichment extracting method includes：The Tris 10mM of guanidinium isothiocyanate 2M~6M, pH8.0,
EDTA 10mM, the Triton X-100 1%~10% (V/V) and SDS 1%~5% (W/V) of pH8.0, solvent is sterilized water.
Eluent 1 in described enrichment extracting method includes：The Tris 100mM of NaCl 0.5M~5M, pH8.0,
The EDTA 1mM and absolute ethyl alcohol 40% (V/V) of pH8.0, solvent is sterilized water.
Eluent 2 in described enrichment extracting method is the ethanol of volumetric concentration 60%~80%.
Digestion treatment fluid in described enrichment extracting method includes：10μL 25mM ATP、25μL Reaction
Buffer, 30-60U PSAD enzymes, deionized water are added to 250 μ L；Described Reaction buffer include：330mM
Tris-acetate, 660mM potassium acetate of pH7.5,100mM magnesium acetates and 5.0mM DTT, solvent is sterilized water.
Eluent 3 in described enrichment extracting method is sterilizing ddH2O or TE buffer solutions.
Another aspect, the invention provides the PCR detection method of one kind detection hepatitis type B virus (HBV) cccDNA,
Described PCR detection method is comprised the following steps：
(1) enrichment of HBV cccDNA is extracted：The enrichment extracting method of as above-mentioned a kind of HBV cccDNA, to be treated
Survey DNA sample；
(2) PCR amplifications：
1) PCR reaction systems are prepared：
PCR reaction systems, 50 μ L systems：
Described PCR reaction solutions contain 15~25mM Tris-HCl (pH8.0), 15~25mM KCl, 2.5~5mM
(NH4)SO4, 2~5mM MgCl2, 0.5~2mM dNTP/UTP, 200~600nM detection primer groups, 100~300nM detection spies
Pin, 200~600nM internal control primer sets, 100~300nM internal control probes；
2) real-time fluorescence quantitative PCR：
Real-time fluorescence quantitative PCR program is：
The first step：37 DEG C, 5 minutes；Second step：95 DEG C, 10 minutes；3rd step：95 DEG C, 15 seconds；62~68 DEG C, 15 seconds；
72 DEG C, 20 seconds；5~15 circulations；4th step：95 DEG C, 1 second；60 DEG C, 1 minute；40 circulations；Fluorescence is gathered at 60 DEG C；
(3) interpretation of result：Standard is made as qualitative reference product using the plasmid of the HBV cccDNA containing concentration known
Curve, the concentration of the HBV cccDNA in DNA sample to be measured is calculated according to standard curve.
Compared with prior art, the invention has the advantages that：
(1) the enrichment extracting method of the HBV cccDNA that the present invention is provided, initiatively by magnetic bead extraction method and PSAD enzymes
Facture is combined, and both reduces linear DNA, turn avoid dilution of the ferment treatment process to sample, while using the suction of magnetic bead
Attached extraction effect enrichment cccDNA, improves specificity and the sensitivity of detection.
(2) eluent provided in the enrichment extracting method of the HBV cccDNA that the present invention is provided can be removed effectively instead
Answer the impurity in system so that the yield and purity of cccDNA are greatly improved.
(3) provided by the present invention for detecting the detection primer group and detection probe of HBV cccDNA for HBV cccDNA
The high specificity of amplification, can effectively improve detection accuracy.
(4) PCR detection method of the HBV cccDNA that the present invention is provided, with hypersensitivity, can detect low concentration
Sample, its lowest detection is limited to 60 copies/mL.
(5) lysate in the enrichment extracting method that the present invention is provided, eluent 1, eluent 2 and digestion treatment fluid for
Configuration is voluntarily researched and developed in invention, different from conventional reagent so that HBV cccDNA DNA purities and efficiency are higher than prior art.
Brief description of the drawings
Fig. 1 is the canonical plotting in embodiment 5.
Fig. 2 is 36 amplification curves of clinical serum sample in embodiment 5.
Fig. 3 is the canonical plotting in embodiment 6.
Specific embodiment
The explanation of following examples is only intended to help and understands the method for the present invention and its core concept.It should be pointed out that right
For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out
Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.To disclosed implementation
The description below of example, enables professional and technical personnel in the field to realize or uses the present invention.Various modifications to these embodiments
Will be apparent for those skilled in the art, generic principles defined herein can not depart from this
In the case of the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can apply to meet the broader model consistent with principles disclosed herein and features of novelty
Enclose.
Unless otherwise defined, all technologies used herein and scientific terminology have and the technical field of the invention
The identical meaning that those of ordinary skill is generally understood that.
A kind of detection primer group and detection probe for detecting HBV cccDNA of embodiment 1
Detection primer group is made up of following 4 primer sequences：Primer 1：Its nucleotide sequence such as SEQ ID NO：Shown in 1；
Primer 2：Its nucleotide sequence such as SEQ ID NO：Shown in 2；Primer 3：Its nucleotide sequence such as SEQ ID NO：Shown in 3；Primer
4：Its nucleotide sequence such as SEQ ID NO：Shown in 4；
Detection probe nucleotides sequence is classified as SEQ ID NO：Nucleotide sequence shown in 9, the nucleotide sequence of detection probe
5 ' ends be marked with fluorophor FAM, the quenching group TAMRA that 3 ' ends of detection probe are marked with.
A kind of detection primer group and detection probe for detecting HBV cccDNA of embodiment 2
Detection primer group is made up of following 4 primer sequences：Primer 5：Its nucleotide sequence such as SEQ ID NO：Shown in 5；
Primer 6：Its nucleotide sequence such as SEQ ID NO：Shown in 6；Primer 7：Its nucleotide sequence such as SEQ ID NO：Shown in 7；Primer
8：Its nucleotide sequence such as SEQ ID NO：Shown in 8；
Detection probe nucleotides sequence is classified as SEQ ID NO：Nucleotide sequence shown in 9, the nucleotide sequence of detection probe
5 ' ends be marked with fluorophor FAM, the quenching group MGB that 3 ' ends of detection probe are marked with.
A kind of kit for detecting HBV cccDNA of embodiment 3
The kit includes the detection primer group and detection probe in embodiment 1；Also include that internal control primer sets and internal control are visited
Pin；
Internal control primer sets include lower two primers：Primer 9：Its nucleotide sequence such as SEQ ID NO：Shown in 10；Primer 10：
Its nucleotide sequence such as SEQ ID NO：Shown in 11；
The sequence of internal control probe is SEQ ID NO：Nucleotide sequence shown in 12, the 5 ' of the nucleotide sequence of internal control probe
End is marked with fluorophor VIC, and the 3 ' of internal control probe hold the quenching group TAMRA being marked with.
A kind of kit for detecting HBV cccDNA of embodiment 4
The kit includes the detection primer group and detection probe in embodiment 2；Also include that internal control primer sets and internal control are visited
Pin；
Internal control primer sets include lower two primers：Primer 9：Its nucleotide sequence such as SEQ ID NO：Shown in 10；Primer 10：
Its nucleotide sequence such as SEQ ID NO：Shown in 11；
The sequence of internal control probe is SEQ ID NO：Nucleotide sequence shown in 12, the 5 ' of the nucleotide sequence of internal control probe
End is marked with fluorophor HEX, and the 3 ' of internal control probe hold the quenching group TAMRA being marked with.
A kind of PCR detection method of detection hepatitis type B virus (HBV) cccDNA of embodiment 5 and its application
First, using the detection kit in embodiment 3
2nd, samples sources
36 HBV infection person's serum samples.
3rd, detection method
(1) enrichment of HBV cccDNA is extracted：
1. sample to be tested is obtained：200 μ L serum samples are taken, is put into centrifuge tube, cracked to 300 μ L are added in centrifuge tube
Liquid, 20 μ L Proteinase Ks and 20 μ L magnetic beads, are vortexed after mixing and are placed in 50~60 DEG C of oscillation incubations 15 minutes, that is, obtain sample to be tested；
Described lysate includes：EDTA 10mM, the Triton X-100 of Tris 10mM, pH8.0 of guanidinium isothiocyanate 2M, pH8.0
1% (V/V) and SDS 1% (W/V), solvent is sterilized water；
2. the sample to be tested that 1. step obtains is transferred on magnetic frame to place and collects within 5-10 minutes magnetic bead, centrifuge tube is abandoned in suction
In supernatant；
3. the collection for 2. being obtained to step has in the centrifuge tube of magnetic bead and adds 500 μ L eluents 1, is vortexed after mixing, will be from
Heart pipe is transferred on magnetic frame to place collects magnetic bead, the supernatant that suction is abandoned in centrifuge tube for 3-5 minutes；Described eluent 1 includes：
The EDTA 1mM and absolute ethyl alcohol 40% (V/V) of Tris 100mM, pH8.0 of NaCl 0.5M, pH8.0, solvent is sterilized water；
4. the collection for 3. being obtained to step adds 500 μ L eluents 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifugation
Pipe is transferred on magnetic frame to place collects magnetic bead, the supernatant that suction is abandoned in centrifuge tube for 3-5 minutes；Described eluent 2 is volume
The ethanol of concentration 60%；
5. the collection for 4. being obtained to step adds 500 μ L eluents 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifugation
Pipe is transferred on magnetic frame to place collects magnetic bead, the supernatant that suction is abandoned in centrifuge tube for 3-5 minutes；Described eluent 2 is volume
The ethanol of concentration 60%；
6. the collection for 5. being obtained to step adds 300 μ L digestion treatment fluids in having the centrifuge tube of magnetic bead, is vortexed after mixing, and 37
DEG C be incubated 1h after, immediately in 70 DEG C be incubated 30min；Described digestion treatment fluid includes：10μL 25mM ATP、25μL
Reaction buffer, 30U PSAD enzymes, deionized water are added to 250 μ L；Described Reaction buffer include：
The DTT of the potassium acetate of Tris-acetate, 660mM of 330mM pH7.5, the magnesium acetate of 100mM and 5.0mM, solvent is aseptic
Water；
7. after the drop on the of short duration centrifugation tube wall being collected by centrifugation after 6. step is incubated, centrifuge tube is transferred to magnetic frame
Upper to place 1 minute, all of raffinate is abandoned in suction, is opened centrifuge tube lid and is dried 5-10 minutes；
8. 30 μ L eluents 3 are added in the dried centrifuge tube for 7. obtaining to step, is vortexed after mixing, stand 3-10 points
Clock, centrifuge tube is transferred on magnetic frame to place 3-5 minutes and collects magnetic bead, separates supernatant；Described eluent 3 is sterilizing
ddH2O；
9. the supernatant that 8. step obtains is transferred in new centrifuge tube, DNA sample as to be measured；
(2) prepared by HBV cccDNA qualitative references product
Plasmid containing HBV cccDNA purpose fragments is calculated into plasmid concentration with OD values determination methods, it is negative with HBV
Serum-dilution to HBV cccDNA contents are respectively 107Copy/mL, 106Copy/mL, 105Copy/mL, 104Copy/mL conducts
Standard items, for drawing standard curve, extraction are synchronized with sample in (1), and HBV cccDNA negative serums are right as feminine gender
Also synchronously extracted according to product；
(3) PCR amplifications：
1) PCR reaction systems are prepared：
PCR reaction systems, 50 μ L systems：
Described PCR reaction solutions contain 15mM Tris-HCl (pH8.0), 15mM KCl, 2.5mM (NH4)SO4, 2mM
MgCl2, 0.5mM dNTP/UTP, 600nM detection primer groups, 300nM detection probes, 200nM internal control primer sets, 100nM internal controls
Probe；The concentration of wherein primer 1-4 is：The concentration of 150nM, primer 9 and primer 10 is 100nM；
2) real-time fluorescence quantitative PCR：
Real-time fluorescence quantitative PCR program is：The first step：37 DEG C, 5 minutes；Second step：95 DEG C, 10 minutes；3rd step：95
DEG C, 15 seconds, 62 DEG C, 15 seconds, 72 DEG C, 20 seconds, 15 circulations；4th step：95 DEG C, 1 second, 60 DEG C, 1 minute, 40 circulations；60℃
When gather fluorescence；
(3) interpretation of result：The testing result of HBV cccDNA negative serums is feminine gender, is drawn using standard items amplification
Standard curve as shown in figure 1, its R2It is 0.999；The amplification curve of 36 clinical serum samples is as shown in Figure 2；It is bent with standard
The concentration of HBV cccDNA in the quantitative serum sample of line, the quantitative result of 36 clinical serum samples is as shown in table 1 below.
The serum sample quantitative result of table 1
Undet.：undetected.
A kind of PCR detection method of detection hepatitis type B virus (HBV) cccDNA of embodiment 6 and its application
First, using the detection kit in embodiment 4
2nd, samples sources
4 HBV infection person's hepatic tissue samples.
3rd, detection method
3rd, detection method
(1) enrichment of HBV cccDNA is extracted：
1. sample to be tested is obtained：20mg hepatic tissue samples are taken, after chopping, is put into centrifuge tube, to adding 300 in centrifuge tube
μ L lysates, 20 μ L Proteinase Ks and 20 μ L magnetic beads, are vortexed and 50~60 DEG C of oscillation incubations are placed in after mixing, and are incubated complete to hepatic tissue
CL；Described lysate includes：EDTA 10mM, Triton of Tris 10mM, pH8.0 of guanidinium isothiocyanate 6M, pH8.0
X-100 10% (V/V) and SDS 5% (W/V), solvent is sterilized water；
2. the sample to be tested that 1. step obtains is transferred on magnetic frame to place and collects within 5-10 minutes magnetic bead, centrifuge tube is abandoned in suction
In supernatant；
3. the collection for 2. being obtained to step has in the centrifuge tube of magnetic bead and adds 500 μ L eluents 1, is vortexed after mixing, will be from
Heart pipe is transferred on magnetic frame to place collects magnetic bead, the supernatant that suction is abandoned in centrifuge tube for 3-5 minutes；Described eluent 1 includes：
The EDTA 1mM and absolute ethyl alcohol 40% (V/V) of Tris 100mM, pH8.0 of NaCl 5M, pH8.0, solvent is sterilized water；
4. the collection for 3. being obtained to step adds 500 μ L eluents 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifugation
Pipe is transferred on magnetic frame to place collects magnetic bead, the supernatant that suction is abandoned in centrifuge tube for 3-5 minutes；Described eluent 2 is volume
The ethanol of concentration 80%；
5. the collection for 4. being obtained to step adds 500 μ L eluents 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifugation
Pipe is transferred on magnetic frame to place collects magnetic bead, the supernatant that suction is abandoned in centrifuge tube for 3-5 minutes；Described eluent 2 is volume
The ethanol of concentration 80%；
6. the collection for 5. being obtained to step adds 300 μ L digestion treatment fluids in having the centrifuge tube of magnetic bead, is vortexed after mixing, and 37
DEG C be incubated 1h after, immediately in 70 DEG C be incubated 30min；Described digestion treatment fluid includes：10μL 25mM ATP、25μL
Reaction buffer, 60U PSAD enzymes, deionized water are added to 250 μ L；Described Reaction buffer include：
The DTT of the potassium acetate of Tris-acetate, 660mM of 330mM pH7.5, the magnesium acetate of 100mM and 5.0mM, solvent is aseptic
Water；
7. after the drop on the of short duration centrifugation tube wall being collected by centrifugation after 6. step is incubated, centrifuge tube is transferred to magnetic frame
Upper to place 1 minute, all of raffinate is abandoned in suction, is opened centrifuge tube lid and is dried 5-10 minutes；
8. 30 μ L eluents 3 are added in the dried centrifuge tube for 7. obtaining to step, is vortexed after mixing, stand 3-10 points
Clock, centrifuge tube is transferred on magnetic frame to place 3-5 minutes and collects magnetic bead, separates supernatant；Described eluent 3 is buffered for TE
Liquid；
9. the supernatant that 8. step obtains is transferred in new centrifuge tube, DNA sample as to be measured；
(2) prepared by HBV cccDNA qualitative references product
Plasmid containing HBV cccDNA purpose fragments is calculated into plasmid concentration with OD values determination methods, TE buffer solutions are used
It is diluted to HBV cccDNA contents and is respectively 106Copy/μ L, 105Copy/μ L, 104Copy/μ L and 103Copy/μ L, conduct mark
Quasi- product, for drawing standard curve, standard items directly enter performing PCR amplification, and TE buffer solutions are also directly carried out as negative controls
PCR is expanded；
(3) PCR amplifications：
1) PCR reaction systems are prepared：
PCR reaction systems, 50 μ L systems：
Described PCR reaction solutions contain 25mM Tris-HCl (pH8.0), 25mM KCl, 5mM (NH4)SO4, 5mM
MgCl2, 2mM dNTP/UTP, 400nM detection primer groups, 100nM detection probes, 200nM internal control primer sets, 100nM internal controls spy
Pin；The concentration of wherein primer 5-8 is：The concentration of 100nM, primer 9 and primer 10 is 100nM；
2) real-time fluorescence quantitative PCR：
Real-time fluorescence quantitative PCR program is：The first step：37 DEG C, 5 minutes；Second step：95 DEG C, 10 minutes；3rd step：95
DEG C, 15 seconds, 66 DEG C, 15 seconds, 72 DEG C, 20 seconds, 15 circulations；4th step：95 DEG C, 1 second, 60 DEG C, 1 minute, 40 circulations；60℃
When gather fluorescence；
(3) interpretation of result：Negative controls show that the standard curve drawn using standard items amplification is such as schemed without CT values
Shown in 3, its R2It is 0.999；The amplification curve of 36 clinical serum samples is as shown in Figure 2；With the quantitative serum sample of standard curve
The concentration of middle HBV cccDNA, with the concentration of HBV cccDNA in the quantitative hepatic tissue DNA sample of standard curve, liver sample HBV
The concentration * 30 μ L/20mg's of HBV cccDNA in cccDNA (copy/mg)=DNA sample, 4 hepatic tissue sample cccDNA determines
Amount result is as shown in table 2 below.
The hepatic tissue sample quantitative result of table 2
| Sample number | 1 | 2 | 3 | 4 |
| Quantitative result (copy/mg) | 3.09E+03 | 6.58E+03 | 7.90E+00 | 2.89E+02 |
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Within god and principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
SEQUENCE LISTING
<110>Guangzhou Supbio Bio-Technology Co., Ltd.
<120>The enrichment extracting method and detection primer group, probe and method of HBV cccDNA
<130> 20170314
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
actccccgtc tgtgccttct 20
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
ttctttatac gggtcaatgt cca 23
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
aggaggctgt aggcataaat 20
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence
<400> 4
tcccgataca aagcagag 18
<210> 5
<211> 24
<212> DNA
<213>Artificial sequence
<400> 5
acgaccgacc ttgaggcata cttc 24
<210> 6
<211> 24
<212> DNA
<213>Artificial sequence
<400> 6
attcatcaac tcaccccaac acag 24
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<211> 21
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<213>Artificial sequence
<400> 7
taggaggctg taggcataaa t 21
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<211> 20
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<213>Artificial sequence
<400> 9
ctgttcaagc ctccaagctg 20
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<211> 29
<212> DNA
<213>Artificial sequence
<400> 10
cggggtcacc cacactgtgc ccatctacg 29
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<400> 11
ggtcacccac actgtgccca tctacg 26
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atgccctccc ccatgccatc ct 22
Claims (10)
1. a kind of detection primer group and detection probe for detecting HBV cccDNA, it is characterised in that：Described detection primer
Group is primer sets 1 or primer sets 2；Described primer sets 1 are by following 4 primer sequences or the complementary sequence of following 4 primer sequences
Row composition：Primer 1：Its nucleotide sequence such as SEQ ID NO：Shown in 1；Primer 2：Its nucleotide sequence such as SEQ ID NO：2 institutes
Show；Primer 3：Its nucleotide sequence such as SEQ ID NO：Shown in 3；Primer 4：Its nucleotide sequence such as SEQ ID NO：Shown in 4；Institute
The primer sets 2 stated are made up of the complementary series of following 4 primer sequences or following 4 primer sequences：Primer 5：Its nucleotides sequence
Row such as SEQ ID NO：Shown in 5；Primer 6：Its nucleotide sequence such as SEQ ID NO：Shown in 6；Primer 7：Its nucleotide sequence is such as
SEQ ID NO：Shown in 7；Primer 8：Its nucleotide sequence such as SEQ ID NO：Shown in 8；
The nucleotides sequence of described detection probe is classified as SEQ ID NO：Sequence or its complementary series shown in 9；Described detection
5 ' ends of the nucleotide sequence of probe are marked with fluorophor, and described fluorophor is in FAM, HEX, VIC, CY5 and TET
It is a kind of；The quenching group that 3 ' ends of described detection probe are marked with, described quenching group is in TAMRA, MGB and BHQ
It is a kind of.
2. detection primer group as claimed in claim 1 and detection probe, it is characterised in that：Described detection primer group is primer
Group 1；Described primer sets 1 are made up of the complementary series of following 4 primer sequences or following 4 primer sequences：Primer 1：Its core
Nucleotide sequence such as SEQ ID NO：Shown in 1；Primer 2：Its nucleotide sequence such as SEQ ID NO：Shown in 2；Primer 3：Its nucleotides
Sequence such as SEQ ID NO：Shown in 3；Primer 4：Its nucleotide sequence such as SEQ ID NO：Shown in 4.
3. detection primer group as claimed in claim 1 and detection probe, it is characterised in that：Described detection primer group is primer
Group 2；Described primer sets 2 are made up of the complementary series of following 4 primer sequences or following 4 primer sequences：Primer 5：Its core
Nucleotide sequence such as SEQ ID NO：Shown in 5；Primer 6：Its nucleotide sequence such as SEQ ID NO：Shown in 6；Primer 7：Its nucleotides
Sequence such as SEQ ID NO：Shown in 7；Primer 8：Its nucleotide sequence such as SEQ ID NO：Shown in 8.
4. a kind of kit for detecting HBV cccDNA, it is characterised in that：Described kit is appointed including claim 1-3
Detection primer group and detection probe described in meaning one.
5. kit as claimed in claim 4, it is characterised in that：Described kit also includes that internal control primer sets and internal control are visited
Pin；Described internal control primer sets include lower two primers：Primer 9：Its nucleotide sequence such as SEQ ID NO：Shown in 10；Primer
10：Its nucleotide sequence such as SEQ ID NO：Shown in 11；The sequence of described internal control probe is SEQ ID NO：Sequence shown in 12
Row；5 ' ends of the nucleotide sequence of described internal control probe are marked with fluorophor, described fluorophor is FAM, HEX,
One kind in VIC, CY5 and TET；3 ' the quenching groups that are marked with of end of described internal control probe, described quenching group is
One kind in TAMRA, MGB and BHQ, the fluorophor on described internal control probe should be different from the fluorescent base in detection probe
Group.
6. a kind of enrichment extracting method of HBV cccDNA, it is characterised in that：Described enrichment extracting method is comprised the following steps：
(1) sample to be tested is obtained：After test serum sample is shredded, it is put into centrifuge tube, to addition lysate, protease in centrifuge tube
K and magnetic bead, are vortexed and 50~60 DEG C of oscillation incubations are placed in after mixing, and incubation is completely dissolved to hepatic tissue, that is, obtain sample to be tested；Such as
Fruit is serum sample：Then take serum sample to be put into centrifuge tube, to lysate, Proteinase K and magnetic bead is added in centrifuge tube, be vortexed
50~60 DEG C of oscillation incubations are placed in after mixing 15 minutes, that is, obtain sample to be tested；(2) sample to be tested for obtaining step (1) turns
To move to placed on magnetic frame and collect within 5-10 minutes magnetic bead, the supernatant that suction is abandoned in centrifuge tube；(3) collection obtained to step (2)
There is addition eluent 1 in the centrifuge tube of magnetic bead, be vortexed after mixing, centrifuge tube is transferred on magnetic frame to place 3-5 minutes and is collected
Magnetic bead, the supernatant that suction is abandoned in centrifuge tube；(4) collection obtained to step (3) adds eluent 2 in having the centrifuge tube of magnetic bead,
After rotation is mixed, centrifuge tube is transferred on magnetic frame to place and collects within 3-5 minutes magnetic bead, the supernatant that suction is abandoned in centrifuge tube；(5) to
The collection that step (4) is obtained adds eluent 2 in having the centrifuge tube of magnetic bead, after rotation mixing, centrifuge tube is transferred on magnetic frame
Place and collect within 3-5 minutes magnetic bead, the supernatant that suction is abandoned in centrifuge tube；(6) collection obtained to step (5) has the centrifuge tube of magnetic bead
Middle addition digestion treatment fluid, is vortexed after mixing, and after 37 DEG C are incubated 1h, is incubated 30min in 70 DEG C immediately；(7) it is of short duration to be collected by centrifugation
After the drop on centrifugation tube wall after step (6) incubation, centrifuge tube is transferred on magnetic frame and is placed 1 minute, suction abandons all
Raffinate, open centrifuge tube lid dry 5-10 minutes；(8) wash-out is added in the dried centrifuge tube for obtaining to step (7)
Liquid 3, is vortexed after mixing, and stands 3-10 minutes, centrifuge tube is transferred on magnetic frame to place 3-5 minutes and collects magnetic bead, in separation
Clear liquid；(9) supernatant that step (8) is obtained is transferred in new centrifuge tube, is stored in -20 DEG C or is directly used in sample-adding.
It is 7. as claimed in claim 6 to be enriched with extracting method, it is characterised in that：Described lysate includes：Guanidinium isothiocyanate 2M
~6M, the EDTA 10mM of Tris 10mM, pH8.0 of pH8.0, Triton X-1001%~10% (V/V) and SDS 1%~
5% (W/V), solvent is sterilized water.
It is 8. as claimed in claim 6 to be enriched with extracting method, it is characterised in that：Described eluent 1 includes：NaCl 0.5M~
The EDTA 1mM and absolute ethyl alcohol 40% (V/V) of Tris 100mM, pH8.0 of 5M, pH8.0, solvent is sterilized water；Described washes
De- liquid 2 is the ethanol of volumetric concentration 60%~80%.
It is 9. as claimed in claim 6 to be enriched with extracting method, it is characterised in that：Described digestion treatment fluid includes：10μL 25mM
ATP, 25 μ L Reaction buffer, 30-60U PSAD enzymes, deionized water are added to 250 μ L；Described Reaction
Buffer includes：Tris-acetate, 660mM potassium acetate of 330mM pH7.5,100mM magnesium acetates and 5.0mM DTT, solvent
It is sterilized water.
10. a kind of PCR detection method of HBVcccDNA, it is characterised in that：Described PCR detection method is comprised the following steps：
(1) enrichment of HBV cccDNA is extracted, to obtain DNA sample to be measured：Step (1) is claim 6-9 any one institute
The enrichment extracting method of the HBV cccDNA for stating；
(2) PCR amplifications：
1) PCR reaction systems are prepared：
PCR reaction systems, 50 μ L systems：
Described PCR reaction solutions contain 15~25mM Tris-HCl (pH8.0), 15~25mM KCl, 2.5~5mM (NH4)SO4,
2~5mM MgCl2, 0.5~2mM dNTP/UTP, 200~600nM detection primer groups, 100~300nM detection probes, 200~
600nM internal control primer sets, 100~300nM internal control probes；
2) real-time fluorescence quantitative PCR：
Real-time fluorescence quantitative PCR program is：
The first step：37 DEG C, 5 minutes；
Second step：95 DEG C, 10 minutes；
3rd step：95 DEG C, 15 seconds；62~68 DEG C, 15 seconds；72 DEG C, 20 seconds；5~15 circulations；
4th step：95 DEG C, 1 second；60 DEG C, 1 minute；40 circulations；Fluorescence is gathered at 60 DEG C；
(3) interpretation of result：Standard curve is made as qualitative reference product using the plasmid of the HBV cccDNA containing concentration known,
The concentration of the HBV cccDNA in DNA sample to be measured is calculated according to standard curve.
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