CN104894258B - PNA clampers real-time quantitative PCR detects HBV linear DNA ratios - Google Patents
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Abstract
The invention discloses a kind of method of PNA clampers real-time quantitative PCR detection HBV linear DNA ratios, include the following steps：(1) HBV DNA profilings are pre-processed with archaeal dna polymerase and endonuclease, produces the different templates of 12 bases of difference；(2) making choice property of dlDNA is expanded using primer pair and peptide nucleic acid, the HBV dlDNA quantitative values and total HBV DNA of (3) in quantitative PCR detection result, are calculated dlDNA ratios.Sensitivity and accuracy are higher, compensate for the sxemiquantitative of the Southern Blot methods of classics, complicated, the limitations such as sensitivity is low.Technical support is provided for HBV dlDNA ratios detection in further detection and analysis slow hepatitis B patients serum.
Description
Technical field
The present invention relates to a kind of related HBV DNA to detect.In particular it is to be related to a kind of detection detection HBV linear DNAs
The method of ratio.In addition, the invention further relates to the kit that the detection method is used.
Background technology
Hepatitis type B virus (Hepatitis B virus, HBV) is hepadnavirus.With infective HBV
Dane's particles include two kinds of genomic forms, partially double stranded loose cyclic DNA (Relaxed-circular DNA, rcDNA)
With double-stranded linear DNA (Duplex-linear DNA, dlDNA).From the generation of both genomic forms is based on when normal chain synthesizes
The difference of beginning primer binding site.It is (the Direct repeat of direct repeat sequence 1 in the original location that the primer synthesized such as normal chain, which stops,
1, DR1), directly extension synthesis normal chain, referred to as original position extend, and the genome of synthesis is dlDNA.If the indexing of positive strand primer is to directly
Synthesizing normal chain again on repetitive sequence 2 (Direct repeat2, DR2), be known as indexing extension, then the genome synthesized is rcDNA,
For the rcDNA of cyclization in addition to than 224 bases of dlDNA long, both remaining base sequences are identical [Fig. 1, bibliography 1].It is previously more
Research report show in Hepadna Virus section dlDNA it is related with the integration of host genome and the generation of liver cancer [bibliography 2,
3].But still lack so far and the large sample of serum HBV dlDNA ratios in slow hepatitis B patient is investigated, its clinical meaning is not
It is bright.
The past studies have shown that serum HBV dlDNA ratios are about the 5-15% of total HBV DNA, but previously to HBV DNA
The analysis of genomic form mainly [is joined using directly observational technique under Electronic Speculum after Southern hybrid methods and viral genome extraction
Examine document 4,5].These methods are not suitable for clinic there are the shortcomings of complicated, detection time is long, sensitivity is low, sxemiquantitative
Sample analysis.In theory, HBV dlDNA ratios can be by expanding total HBV DNA (dlDNA and rcDNA) and individually amplification
RcDNA methods are calculated, and this method needs to design two pairs of primers, and a pair is used for expanding total HBV DNA, and pair of primers expands
Increase rcDNA.But the difference between different primers sequence with amplification length, cause their melting temperature, joint efficiency, expansion
The differences such as Increasing Efficiency, add the uncertainty and incommensurability of experiment.Therefore, it is slow there is an urgent need for establishing easy reliable detection at present
The method of hepatitis B infected person's serum HBV dlDNA ratios, to inquire into its clinical value.
Based on the difference of HBV rcDNA and dlDNA genome structures, we devise PNA clampers qPCR to detect serum
Middle HBV dlDNA ratios.Peptide nucleic acid (PNA) is a kind of analogies of oligonucleotides, its classical molecular structure is by repeatability
N- (2- amino-ethyls) glycine units by amido link be connected substitution DNA molecular in phosphopentose skeleton formed one
The analog [bibliography 6] of kind DNA molecular.Although pna molecule skeleton and DNA molecular are dramatically different, PNA and complementary nucleic acid
Between combination still follow the principle of base pair complementarity, and since PNA is in electroneutral, it has higher than natural nucleotide
Affinity and specificity.Although PNA's can specifically bind with DNA profiling, template can not be prolonged as PCR primer
Stretch.Therefore PNA and PCR primer can be combined competitively with DNA profiling and specificity suppression PCR reacts [bibliography 7].I
Have evaluated PNA clampers real-time quantitative PCR detection HBVdlDNA ratios sensitivity, specificity and accuracy.Thereby produce
The present invention.
The content of the invention
The object of the present invention is to provide a kind of method of PNA clampers real-time quantitative PCR detection HBV linear DNA ratios.This hair
Bright another object is to provide a kind of kit for the detection method.
The present invention is adopted the following technical scheme that to realize the above-mentioned purpose of the present invention.
The method of PNA clampers real-time quantitative PCR detection HBV linear DNA ratios of the present invention, includes the following steps：
(1) HBV DNA profilings are pre-processed with archaeal dna polymerase and endonuclease, produces the different templates of 12 bases of difference；
(2) making choice property of dlDNA is expanded using primer pair and peptide nucleic acid；(3) HBV in quantitative PCR detection result
DlDNA quantitative values and total HBV DNA, are calculated dlDNA ratios.
According to our result of the test, restriction endonuclease contributes to the reparation of archaeal dna polymerase.It is therefore preferable that side
Case is to use the pretreatment of archaeal dna polymerase and restriction endonuclease to HBV DNA profilings at the same time in step (1).It is described
Archaeal dna polymerase and the preferred klenow archaeal dna polymerases of restriction endonuclease and Hpy8 I restriction endonucleases.
Structure sequence feature based on rcDNA and dlDNA, we devise PNA clamper qPCR technologies, using pair of primers
With a PNA, total HBV DNA ratios are accounted for detect dlDNA, ensure that the specificity and accuracy of this method.This method
Key Design is to use pretreatment of restriction endonuclease Hyp8 I and the Klenow archaeal dna polymerase to HBV DNA profilings,
The different templates of 12 bases of difference, i.e. the rcDNA templates of 240bp and the dlDNA moulds of 228bp are generated in 5 ' ends of normal chain
Plate.This species diversity becomes us and designs the basis of the suppression rcDNA amplifications of PNA selectivity.
According to HBV primers and PNA sequences, sequence and relevant position are shown in Table 1-1.
Table 1-1. primer sequences and PNA sequences
Using the primer pair and PNA of design, PNA can be made to be matched with rcDNA complete complementaries and combined, and only number of base
With dlDNA complementary pairings.Therefore, under the annealing conditions of optimization, can make PNA can only with rcDNA with reference to and cannot be with dlDNA
With reference to so as to fulfill the suppression expanded to rcDNA, i.e. selective amplification dlDNA.With optimal conditions, it is possible to achieve the party
The detection sensitivity of method is 0.5%, and the range of linearity is 0.5% to 99%.PNA clamper qPCR methods and Southern-Blot methods
Results contrast, further demonstrates the accuracy of this method.
The present invention also provides a kind of kit, and HBV linear DNA ratios are detected for PNA clampers real-time quantitative PCR, including
Primer pair, peptide nucleic acid and Hpy8 I endonucleases and Klenow polymerases, the primer pair, peptide nucleic acid such as table 1-1 determine
Justice.
The detection HBV dlDNA sensitivity of PNA clamper qPCR methods and accuracy of the present invention is higher, compensate for classics
The sxemiquantitative of Southern-Blot methods, complicated, the limitations such as sensitivity is low, therefore, the new method are established as further
The detection of HBV dlDNA ratios provides technical support in detection and analysis slow hepatitis B patients serum.
Brief description of the drawings
The reproduction process of Fig. 1 HBV DNA
Fig. 2 PNA clampers qPCR detects the design principle of dlDNA ratio methods.(A) rcDNA and dlDNA are located in advance
Reason.(B) the PNA clamper qPCR principles of selective amplification dlDNA.(C) PNA clampers PCR amplification optimal conditions.
The specificity of Fig. 3 PNA clampers qPCR detections dlDNA and range of linearity analysis.(A) concentration of rcDNA and dlDNA with
Copy number/milliliter represents that X-axis represents the analogies being serially diluted.(B) with PNA clampers qPCR detection dlDNA in the mixture
Ratio, informal voucher show dlDNA ratios theoretical value in the mixture, and lath is PNA clamper qPCR detected values.
The accuracy of Fig. 4 dlDNA ratios in Southern hybrid methods verification PNA clampers qPCR detection serum.Left figure is shown
Show, after the repairing of endogenous polymerase, BmgBI and AseI restriction endonucleases carry out digestion to template and produce 1030bp's HBV DNA
The dlDNA fragments of rcDNA fragments and 817bp.It is the testing result of southern hybrid methods in right figure, is PNA clampers under right figure
The testing result of qPCR.N/A represents that southern hybrid methods are not detected by signal.
The functional assessment of klenow archaeal dna polymerases and Hpy8 I restriction endonucleases in Fig. 5 PNA clampers qPCR.(A) warp or not
HBV DNA profilings through the pre- digestions of Hpy8 I, obtained dlDNA ratios are repaired by the Klenow enzymes of different time；(B) will
PUC18-HBV1.2-wt and the pUC18-HBV1.2-T1804A plasmids of (C) Hpy8 I restriction enzyme sites mutation, transfection HepG 2 cell
Afterwards, supernatant is respectively classified into 4 groups, Klenow+Hpy8 I coprocessing group, Klenow enzymatic treatments group, Hpy8 I restriction endonucleases treatment groups and
Control group.The result of dlDNA ratios in PNA clampers qPCR detection supernatants.
Embodiment
The design and synthesis of 1 primer sequence of embodiment and peptide nucleic acid sequence
Primer sequence and PNA sequences are the sequence designs with reference to pUC18-HBV1.2 (GenBank AY040627).
(1) design of primers：
Primer is designed according to HBV sequences (GenBank AY040627), relevant position is shown in Table 1-1, is given birth to by Beijing SBS Genetech
Company of thing Technology Co., Ltd. synthesizes.
(2) peptide nucleic acid：
PNA sequences are designed according to HBV sequences (GenBank AY040627), relevant position is shown in Table 1-1, by South Korea
PANAgene companies synthesize.
The conservative Analysis of 2 PNA of embodiment, primer and restriction enzyme site sequence
According to the sequence of pUC18-HBV1.2 plasmids, the PNA base sequences and corresponding primer sequence difference that we design
For 5 '-CAGCACCATGCAACTTTTTC-3'(1806-1825nt) and 5'-CAACTTTTTCACCTCTG-3'(1816-
1832nt), the restriction enzyme site of restriction endonuclease Hpy8 I is in 1804nt.
The nucleic acid sequence 1804nt-1832nt of HBV DNA includes PNA, forward direction primer, endonuclease Hpy8I restriction enzyme sites,
Therefore the conservative of this 29 bases and specificity are also that experimental design is successfully crucial.Since the region polymorphism may influence
PNA clamper qPCR accuracys, therefore, for totally 29 bases of the 1804-1832nt sequences comprising PNA, primer and restriction enzyme site
We have carried out the conservative Analysis of the section with blast program on pubmed to HBV Strain in GenBank.
In GenBank databases, analyzed by Blast to PNA, forward direction primer binding site and Hpy8 I restriction enzyme sites
Totally 29 bases carry out polymorphism analysis to sequence, that is, 1804-1832nt, the results show that in 20000 HBV DNA virus of detection
In strain, there are the sequence of 1-5 base mutation to account for 4.43% (886/20000) for this 29 bases, in this 886 sequences,
65% Strain is Gene A type, and 20% viral pnca gene is D and F types, and the ratio that gene B and c-type account for<10%.
According to the Morbidity investigation of nearest China HBV DNA genotype the results show that A genotype accounts for 0.5%, 1 B gene type
Account for 38.6%, C genotype and account for about 57%, 1 B gene type and C genotype are absolute predominance genotype, and proportion is up to
95.6%.Therefore, the present invention can be widely used in HBV dlDNA proportional levels in detection China slow hepatitis B patients serum.
This also prompts us, by the way that 1804nt-1832nt sequence sections are sequenced, contribute to the detected values of dlDNA ratios into
Row verification.
3 PNA clampers real-time quantitative PCR of embodiment detects HBV linear DNAs
In HBV DNA genomes, rcDNA is held more than dlDNA in addition to 224 bases except the 5 ' of normal chain, remaining sequence
The two identical [bibliography 1].Based on the difference in structure and sequence, we devise PNA clampers qPCR selectivity
Expand dlDNA.
First, we use the HBV DNA moulds of Klenow archaeal dna polymerases and restriction endonuclease Hpy8 I to extraction
Plate carries out digestion and repairing.RcDNA produces one section from 1804nt to 2043nt after restriction endonuclease Hpy8 I digestions
The fragment of totally 240 bases, and there are the discontinuous fragments of 12bp at the 3 ' of this fragment minus strand ends.It is discontinuous negative in order to repair this
The end of chain 3 ', we to the 45 DEG C of denaturation of digestion rear pattern plate, make and the discontinuous minus strand fragments of the 12bp of the termini-complementary of normal chain 5 ' first
Dissociate to get off from the normal chain template, produce a 3 ' end gaps, Klenow archaeal dna polymerases then can be with indentation, there minus strand
3 ' ends combine, using normal chain as template, extend minus strand polishing notch, produce the complete double-strands of 240bp " rcDNA " template.Together
When since the initiation site of dlDNA is 1816nt, after Hpy8 I enzyme digestions, produce one section and be total to from 1816nt to 2043nt
The double chain DNA fragment of 228 bases, this fragment are " dlDNA " template.Secondly, we devise one section and forward direction primer P1F
PNA (1806-1825nt) sequence that (1816-1832nt) overlaps.This design can make PNA and rcDNA complete complementaries
Pairing combines, and only number of base and dlDNA complementary pairings.Therefore, under the annealing conditions of optimization, it can make PNA can only
With rcDNA with reference to and cannot be combined with dlDNA, so as to fulfill the suppression expanded to rcDNA.For this reason, we are in standard PCR protocol
In, the temperature of additional step PNA annealing, makes it prior to forward direction primer annealing combination rcDNA, expansions of the blocking PCR to rcDNA
Increase.
Specifically illustrated below by patient's sample analysis complete procedure.
1st, in 200 μ l serum specimens HBV DNA profilings press Tiangeng biochemical technology Co., Ltd viral DNA extracts kit
Specification operation extraction.It is dissolved in 20 μ l distilled water.
2nd, HBV DNA profilings are handled according to the specification of Hpy8I and KlenowDNA polymerases, operating procedure is such as
Under：
According to the form below is equipped with the 10 μ l of reaction system of digestion and repairing in the EP of 1.5ml on ice：
After complete, brief centrifugation, collects liquid to tube bottom, is incubated at 37 DEG C 15 minutes, 45 DEG C are incubated 15 minutes.
3rd, the real-time quantitative PCR of PNA clampers
Treated HBV DNA profilings are taken to carry out real-time quantitative PCR, system configurations are as follows：
According to the form below prepares PCR reaction solution on ice：
Each standard items and sample is corresponding two groups, are not added with PNA groups and add PNA groups, finally plus a blank control group.
After above-mentioned system is prepared, add in glass capillary, be put into low-temperature and high-speed centrifuge 4500rpm/ minutes, centrifuge 2 minutes,
Then glass capillary is put on fluorescence quantitative PCR instrument.The flow for setting PCR is as follows：95 DEG C of pre-degenerations 15 minutes, 95 DEG C 10
Second, 64 DEG C 15 seconds, 52 DEG C 15 seconds, 72 DEG C 30 seconds, the circulation of coreaction 40.
Optimal conditions：The final concentration of 500nM of PNA, the final concentration of 250nM of PCR primer, 95 DEG C of pre-degenerations 15 minutes,
95 DEG C are denatured 10 seconds, and preferentially annealing 15 seconds, 52 DEG C of PCR primers anneal 15 seconds (Fig. 2 .C) to 64 DEG C of PNA.On this condition, PNA can
To suppress the amplification of rcDNA at 200 times or so, and the amplification on dlDNA does not influence (Fig. 3 .A).Therefore, the detection of this method
Sensitivity is 0.5%, since serum HBV dlDNA ratios are far above 0.5%, sensitivity in this approach be enough to be applied to
Detect HBV dlDNA ratios in clinical serum sample.
By the HBV dlDNA quantitative values (adding PNA groups) in quantitative PCR detection result and total HBV DNA (no PNA groups) phase
Remove, obtain dlDNA ratios.
In order to detect the range of linearity of this method, we are mixed the analogies of dlDNA and rcDNA by different known proportions
Close, then detected with this method.The results show that the testing result of PNA clampers qPCR is consistent with theoretical value (Fig. 3 .B).
4 PNA clampers qPCR of embodiment detects the Accuracy Verification of dlDNA ratios
In order to further verify the accuracy of this method, we pick higher slow of 8 clinical detection HBV DNA levels
Serum of Patients with Hepatitis B sample, according to the method detection wherein HBV dlDNA ratios described in embodiment 3, and with it is classical
Southern hybridization analysis compares.Since HBV gene group normal chain is incomplete DNA chain different in size length 50-90%,
Therefore we carry out endogenous repairing and digestion to HBV DNA, and quantitative comparison is carried out to obtain clear band.
As shown in figure 4, carrying out quantitative analysis to Southern bands gray value with Quantity one softwares, as a result show
Show, PNA clamper qPCR methods are consistent with the result of Southern hybrid methods.
The functional assessment of Klenow polymerases and Hpy8 I restriction endonucleases in 5 PNA clampers qPCR of embodiment
In order to assess the function of Hpy8 I restriction endonucleases, in the case of presence or absence of Hpy8 I restriction endonucleases, Wo Menfen
Klenow polymerase repair times are not determined.Result of the test is shown, is not handled in DNA profiling with Hpy8 I restriction endonucleases
In the case of, it is necessary to Klenow polymerization enzymatic treatments 30 minutes, can just repair HBV rcDNA completely；But if in DNA
In the case that template is handled with Hpy8 I restriction endonucleases, then it polymerize enzymatic treatment 1 minute with Klenow, it is possible to repair completely
HBV rcDNA.Therefore, result of the test shows, Hpy8 I greatly improve the efficiency (Fig. 5 A) of repairing.
In order to assess the effect of Klenow enzymes repairing, we apply pUC18-HBV1.2-WT the and Hpy8 I enzymes of wild type
Cultivated 3 days after the pUC18-HBV1.2-T1804A transfection HepG 2 cells of enzyme site mutation, HBV dlDNA ratios are used in supernatant
PNA clampers qPCR is detected.Then the template of the two is divided into four groups and carries out different disposal i.e. respectively：1) at Klenow+Hpy8 I
Reason group, 2) Klenow enzyme repairing groups, 3) Hpy8 I inscribe digestion groups, 4) control group (without the two processing).As a result such as Fig. 5 B/C
It is shown.In the culture supernatant of pUC18-HBV1.2, pUC18-HBV1.2-T1804A transfection HepG 2 cell, control group HBV
DlDNA ratios are significantly higher than Hpy8 I digestions and Klenow enzyme repairing groups.However, sample is repaiied through Hpy8 I digestions and Klenow enzymes
Benefit group result and the testing result of Klenow polymerase repairing groups do not have significant difference.This shows Klenow polymerases to HBV
The repairing of DNA profiling is must link.But in the case of Klenow polymerases are fully repaired (more than 30 minutes), Hyp8 I inscribes
The digestion of enzyme is not necessarily.
Bibliography
[1].Seeger,C.and W.S.Mason,Hepatitis B virus biology.Microbiol Mol
Biol Rev,2000.64(1):p.51-68.
[2].Toh,S.T.,et al.,Deep sequencing of the hepatitis B virus in
hepatocellular carcinoma patients reveals enriched integration events,
structural alterations and sequence variations.Carcinogenesis,2013.34(4):
p.787-98.
[3].Sung,W.K.,et al.,Genome-wide survey of recurrent HBV integration
in hepatocellular carcinoma.Nat Genet,2012.44(7):p.765-9.
[4].Yamada,M.,et al.,Three distinct Southern blot hybridization
patterns of HBV-DNA in the sera of HBV carriers.Tohoku J Exp Med,1993.170(4):
p.219-28.
[5].Delius,H.,et al.,Structure of the hepatitis B virus genome.J
Virol,1983.47(2):p.337-43.
[6].Egholm,M.,et al.,PNA hybridizes to complementary oligonucleotides
obeying the Watson-Crick hydrogen-bonding rules.Nature,1993.365(6446):p.566-8
[7].Buchardt,O.,et al.,Peptide nucleic acids and their potential
applications in biotechnology.Trends Biotechnol,1993.11(9):p.384-6
Claims (6)
1. a kind of method of PNA clampers real-time quantitative PCR detection HBV linear DNA ratios, includes the following steps：(1) gathered with DNA
Synthase and Hpy8 I endonucleases pre-process HBV DNA profilings, produce the different templates of 12 bases of difference；(2)
Making choice property of dlDNA is expanded using primer pair and peptide nucleic acid；(3) the HBV dlDNA in quantitative PCR detection result determine
Value and total HBV DNA, are calculated dlDNA ratios, wherein the primer and PNA sequences and relevant position are shown in Table 1-1.
2. according to the method described in claim 1, at the same time using in archaeal dna polymerase and Hpy8 I nucleic acid wherein in step (1)
Pretreatment of the enzyme cutting to HBV DNA profilings.
3. according to the method described in claim 2, wherein described archaeal dna polymerase refers to klenow archaeal dna polymerases.
4. according to the method described in claim 1, the flow of setting PCR is as follows wherein in step (2)：95 DEG C of pre-degenerations 15 are divided
Clock, 95 DEG C 10 seconds, 64 DEG C 15 seconds, 52 DEG C 15 seconds, 72 DEG C 30 seconds, the circulation of coreaction 40.
5. according to the method described in claim 1, wherein in step (2), optimal conditions are：The final concentration of 500nM of PNA,
The final concentration of 250nM of PCR primer, 95 DEG C of pre-degenerations 15 minutes, 95 DEG C are denatured 10 seconds, 64 DEG C of PNA preferentially annealing 15 seconds, 52 DEG C
PCR primer is annealed 15 seconds.
6. a kind of kit, for PNA clampers real-time quantitative PCR detect HBV linear DNA ratios, including primer pair, peptide nucleic acid with
And Hpy8 I endonucleases and Klenow polymerases, the primer pair, peptide nucleic acid such as table 1-1 are defined.
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| 肽核酸与核酸分子杂交的模式及其在基因诊断领域中的应用;陈鸣等;《生命的化学》;20031231;第23卷(第5期);第353-355页 * |
| 肽核酸钳制PCR早期检测乙型肝炎病毒YMDD耐药变异;张迎迎等;《J South Med Univ》;20130613;第33卷(第6期);第853-856页 * |
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