CN102534047A - Kit for directly observing distribution and location of hepatitis B virus (HBV) cccDNA in liver tissue - Google Patents
Kit for directly observing distribution and location of hepatitis B virus (HBV) cccDNA in liver tissue Download PDFInfo
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Abstract
The invention relates to a method and a kit for detecting hepatitis B virus cccDNA in situ. The method comprises the following steps of: fixing a sample section of a liver tissue; digesting; performing in-situ rolling cycle and fluorescence-quantitation polymerase chain reaction (PCR) amplification; and detecting an amplification product. By the method, the distribution and location of the hepatitis B virus (HBV) cccDNA in the liver tissue can be directly observed, the operation is simple and convenient, the liver tissue is not required to be made into homogenate, nucleic acid is not required to be extracted to serve as a template, and judgment results are not required to be made into a standard curve.
Description
Technical field:
The present invention relates to the method for a kind of in situ detection hepatitis B virus cccDNA, a kind of direct viewing HBVcccDNA distributes in hepatic tissue and localized method and test kit but particularly relate to.
Background technology:
The detection of hepatitis b virus covalence closed cyclic DNA (covalently closed circular DNA is designated hereinafter simply as HBVcccDNA) is significant aspect the pathogeny of virus disease and antiviral therapy research.
At present, have document to adopt sleeve type PCR method in situ detection HBVcccDNA in hepatic tissue to express, this method can't be got rid of non-closed hoop DNA and the interference of integrating HBV DNA.
Round pcr can be in reaction tubes be used for the nucleic acid amplification of trace for millions of times to analyze, but can not reflect the relation between amplified production and the weave construction.Though hybridization in situ technique can reflect the relation between amplified production and the weave construction, the segmental copy number of purpose is less than under 10 the situation in each cell, and the susceptibility of this method obviously descends.
The original position round pcr combines round pcr and hybridization in situ technique, is direct original position amplification target DNA fragment on the cell or tissue sample, and detects the technology of its amplified production in position.Nineteen ninety Haase etc. has at first reported the original position PCR experiment of a success, and many subsequently laboratories have had report in succession.Mainly be divided into direct in-situ and indirect in situ PCR. by the detection mode difference
Direct in-situ PCR is to use primer or the free nucleotide of mark to carry out original position PCR reaction, and this tagged molecule gets in the amplified production subsequently, but amplification direct viewing and need not carry out in situ hybridization.That direct in-situ PCR has is easy and simple to handle, flow process short, the timesaving advantage.But direct in-situ PCR is prone to primer mispairing or non-specific annealing take place, and occurs false positive easily; In addition, the primer of mark also can reduce PCR efficient.
Indirect in situ PCR is not having to carry out the PCR reaction under the situation of affinity tag, after amplified reaction finishes, detects the signal of amplification again with hybridization in situ technique.This method can overcome because the non-specific problem that DNA repairs or the primer mispairing causes.This method need be carried out wash-out and the in situ hybridization process behind the amplified reaction, thereby required time is longer relatively.(rolling cycle amplification RCA) is a kind of external isothermal nucleic acid amplification method to rolling circle amplification.Principle is an Oligonucleolide primers with after circular template combines, cyclic amplification under the effect of special polysaccharase.
It is the previous work of the inventor that one Chinese patent application 20101023T281.4 " detects paraffin embedding hepatic tissue hepatitis B virus cccDNA "; Adopt rolling circle amplification to add the method detection by quantitative HBV cccDNA that strides breach real-time fluorescence PCR technology for detection closed hoop DNA, solved the bottleneck of restriction HBV cccDNA detection by quantitative preferably.
Though; This method and the conventional compared with techniques of using such as fluorescent probe detection by quantitative can improve detection reaction specificity and sensitivity simultaneously, and with only compare with the rolling circle amplification method; Can carry out HBV cccDNA detection by quantitative, but still exist following shortcoming with not enough:
The one, the detection method more complicated must earlier be processed homogenate to hepatic tissue, and cytoclasis, therefrom extracting goes out nucleic acid and does template, in reaction tubes, carries out detection by quantitative with quantitative real time PCR Instrument then.The result judges need manufacture typical curve, with β-actin as internal reference.
In addition; This method is to be used for the specificity method of HBVcccDNA in the detection by quantitative hepatic tissue; But can not the distribution of HBVcccDNA and hepatic tissue form or histopathologic characteristics be interrelated; The relation that can not reflect HBVcccDNA and hepatic tissue structure can not be observed distribution and the location of HBVcccDNA in liver cell.
Summary of the invention:
The purpose of this invention is to provide and a kind ofly can observe directly distribution and the localized original position HBV cccDNA detection method of HBV cccDNA in hepatic tissue, the relation of reflection HBVcccDNA and hepatic tissue structure at the microscopically naked eyes.But another object of the present invention is distribution and the positioned detection test kit of exploitation direct viewing HBVcccDNA in hepatic tissue.
The technical scheme of present method is:
The detection method of a kind of direct viewing HBVcccDNA Distribution and localization in hepatic tissue, step is: the patients with hepatic tissue samples is fixed, and pcr amplification is carried out in digestion then, detects amplified production at last; It is characterized in that:
1) said sample fixedly is through 10% formaldehyde fixed, paraffin embedding, serial section with the hepatic tissue sample;
2) said digestion is to digest with proteolytic enzyme and PSAD enzyme;
3) said pcr amplification is on hepatic tissue section, to carry out original position to roll ring and add and stride the breach pcr amplification;
4) detection of amplified production is after rolling the anti digoxin antibody that ring adds dropping alkali phosphorus enzyme labelling on the hepatic tissue section after striding the breach pcr amplification, hatching certain hour, drips NBT/BCIP; Naked eyes direct viewing hepatic tissue section colour developing situation under the room temperature simple microscope, it is that HBVcccDNA expresses the positive that blueness, purple and hyacinthine particle person appear in nucleus, it is negative for HBVcccDNA expresses that red person appears in nucleus;
5) whole testing process is all carried out being loaded with on the glass slide of hepatic tissue section.
Said hepatic tissue sample fixed method is: with hepatic tissue sample warp 10% formaldehyde fixed, and paraffin embedding, serial section, after the YLENE dewaxing, gradient ethanol dehydration, drying at room temperature.
Said digestion is to adopt the protease digestion hepatic tissue section to increase tissue permeability earlier, adopts non-covalent closed hoop DNA in the PSAD enzymic digestion tissue then.
The said original position of carrying out is rolled ring and is added that to stride the used instrument of breach pcr amplification be original position PCR appearance.
Carrying out original position rolls ring and adds that to stride the breach pcr amplification be on hepatic tissue section, to add the rolling circle amplification primer and stride the breach primer; Roll to encircle to add and stride breach original position pcr amplification HBVcccDNA, said rolling circle amplification primer is RCA1, RCA2, RCA3, RCA4, RCA5, RCA6, RCA7, RCA8, P59, ten primers of P83 (sequence of primer is seen embodiment 1) with striding the breach primer.
Said adding rolling circle amplification primer is to add said ten primers with striding the breach primer, each primer 0.8 μ l, and at 95 ℃ of insulation 3min, 55 ℃ of insulation 15s, 33 ℃ of insulation 15s, 20 ℃ of insulation 10min; Again above-mentioned each primer is respectively got 0.8 μ l, add Phi29DNA polysaccharase 1 μ l, Phi29 buffer 1 μ l, BSA 0.2 μ 1, dNTP 1.6 μ l, aseptic deionized water 4.2 μ l were added drop-wise in the tissue, 30 ℃ of insulations 18 hours.
The detection of said amplified production is the anti digoxin antibody that on tissue slice, drips alkali phosphatase enzyme mark, hatches 1 * TBS washing for 37 ℃; Drip NBT/BCIP, the dark place color development at room temperature; 0.1% nuclear fast red is redyed karyon, and neutral gum mounting after the drying at room temperature, microscopically are observed and the record result.
If the expression of HBV cccDNA is arranged in the hepatic tissue; Then through original position roll the ring add stride the breach pcr amplification after; On tissue slice, drip the anti digoxin antibody of alkali phosphatase enzyme mark, this antibody will combine with the digoxin in the amplified production, again with tissue slice on the NBT/BCIP that drips react.Principle is: BCIP+NBT (NBT) is one of best substrate combination of SEAP; At SEAP (Alkaline phosphatase; AP) effect down; BCIP can be hydrolyzed the product that produces strong reactivity, and this product meeting and NBT react, and form insoluble mazarine to hepatic NBT-formazan.
Because SEAP and NBT/BCIP reaction produces blue, blue-purple granule, adds the coker fast red and redyes karyon, not only can observe directly distribution and the location of HBV cccDNA in hepatic tissue at microscopically, the pathological change that the while can also tissues observed.
It is a kind of use that is intended to through nuclear fast red dyestuff that the nuclear fast red is redyed reagent, makes nucleus present incarnadine, thereby produces heterochromia and contrast with sample dyeing in the histological chemistry, is convenient to observe.
The reagent and the test kit that can prepare hepatic tissue in situ detection hepatitis B virus cccDNA with method of the present invention.
But distribution and the positioned detection test kit of a kind of direct viewing HBVcccDNA in hepatic tissue; Contain stain remover, dNTP and 1 * PBS, it is characterized in that also containing following one or more materials: tissue slice fixing agent, deparaffinization reagents, closed reagent, proteolytic enzyme, PSAD enzyme, Phi29 archaeal dna polymerase, roll ring primer and digoxigenin labeled primer.
Preferably, the tissue slice fixing agent is 10% formaldehyde; Proteolytic enzyme is Proteinase K, and deparaffinization reagents is a YLENE, and closed reagent is that BSA, stain remover are TritonX-100.
Anti digoxin antibody, 1 * TBS, NBT/BCIP, the nuclear fast red that can also contain Phi29 buffer, alkali phosphatase enzyme mark in the test kit.
The detection method of mentioned reagent box is that the hepatic tissue sample is fixed, and paraffin embedding digests, and carries out original position then and rolls ring and add and stride the breach pcr amplification, at last in microscopically naked eyes direct viewing hepatic tissue section color.
The invention has the beneficial effects as follows:
1, observes directly distribution and the location of HBV cccDNA in hepatic tissue
Help to study the pathogeny and the pathologic process of virus disease.Especially aspect the relation of research virus and tumour, bringing into play important effect.
2, method is easier, need hepatic tissue not processed homogenate, and extracting goes out nucleic acid and does template; Result of determination need not manufactured typical curve yet.
3, highly sensitive
When the hepatitis B patient viral level is lower, in the time of can't detecting with existing hybridization in situ technique, still can detect HBV cccDNA in the hepatic tissue with present method.
4, high specificity
Through the PSAD enzymic digestion, remove the interference of non-covalent closed hoop DNA, carry out rolling circle amplification on this basis and add and stride breach PCR, further remove the PSAD enzyme and do not digest non-completely closed hoop DNA and dna integration, further strengthened specificity.
5, repeatability, good stability
The experimental result susceptible of proof that provides from embodiment originally carries out duplicate detection 3 times to 10 increments in clinical source, and group difference does not have significance (p＜0.05) in the group.
Description of drawings:
Fig. 1 is that different detection methods detect HBV cccDNA comparison diagram in the chronic hepatitis B patient hepatic tissue, wherein
A: method of in situ PCR detects HBV cccDNA in the chronic hepatitis B patient hepatic tissue;
B:PSAD digests+strides breach original position PCR method and detects HBV cccDNA in the chronic hepatitis B patient hepatic tissue;
C: PSAD digestion+RCA+ of the present invention strides breach original position PCR method and detects HBV cccDNA in the chronic hepatitis B patient hepatic tissue;
Fig. 2 is that different detection methods detect HBV cccDNA in the hbv-liver cirrhosis patients with hepatic tissue, wherein
A: method of in situ PCR detects HBV cccDNA in the hbv-liver cirrhosis patients with hepatic tissue;
B:PSAD digests+strides breach original position PCR method and detects HBV cccDNA in the hbv-liver cirrhosis patients with hepatic tissue;
C: PSAD digestion+RCA+ of the present invention strides breach original position PCR method and detects HBVcccDNA in the hbv-liver cirrhosis patients with hepatic tissue;
Fig. 3 is that different detection methods detect HBV ccc DNA in the liver cancer patient hepatic tissue, wherein
A: method of in situ PCR detects HBV cccDNA in the liver cancer patient hepatic tissue;
B:PSAD digests+strides breach original position PCR method and detects HBV cccDNA in the liver cancer patient hepatic tissue;
C: PSAD digestion+RCA+ of the present invention strides breach original position PCR method and detects HBVcccDNA in the liver cancer patient hepatic tissue;
Fig. 4 is a negative control, promptly adopts the inventive method to detect HBV cccDNA in hepatitis C patients, healthy subjects, the transgenic mice hepatic tissue; Set up no primer, no digoxigenin labeled primer, no TaqDNA polymeric enzyme reaction system as negative control respectively to hepatitis B liver cancer patient hepatic tissue.
Embodiment
PSAD enzyme of mentioning in following examples and Phi29 archaeal dna polymerase are respectively available from Epicentre and New England Biolab, and it is synthetic by Shanghai biotechnology ltd to roll the ring primer.
The detection method of embodiment 1 direct viewing HBVcccDNA Distribution and localization in hepatic tissue
One, research sample
Research object is 50 routine chronic type b livers during 302 hospitals of PLA in March, 2010～2011 year Mays, liver cirrhosis, hepatocellular carcinoma patient, wherein male 34 examples, women 16 examples, the range of age 7～68 years old.Diagnosis meets the viral hepatitis Case definition of 2000 the tenth time national viral hepatitis meeting revision, gets rid of first, the third, fourth, penta, hepatitis G virus and blood transfusion transmitted virus, cytomegalovirus, ebv infection and alcohol property, medicine property, autoimmune liver disease.Other collects non-hepatitis B patient hepatic tissue sample as negative control.
Two, method
1, the processing of tissue slice
Put into 70 ℃ of 10% formaldehyde fixed liquid (85% absolute ethyl alcohol, 10% formaldehyde, 5% Glacial acetic acid min. 99.5), 20 minutes wear tissue from patient's liver.Gradient ethanolic soln dehydration: 70 ℃ of 20 minutes → absolute ethyl alcohol II70 of 70 ℃ of 20 minutes → 95% ethanol II70 ℃ of 20 minutes → absolute ethyl alcohol I of 95% ethanol I ℃ 20 fens ethanol; Transparent 20 minutes of YLENE; Paraffin waxdip 30 minutes; The embedding of embedding frame.Hepatic tissue section.For preventing that tissue from coming off and PCR reacted constituent penetrate tissue is arrived in the liver cell nuclear, and combine with target gene fully, at first paraffin-embedded hepatic tissue is cut into 5 μ m, be affixed on the slide glass of crossing with the APES siliconizing, 60 ℃ are toasted 2h.Tissue slice is by extremely washing of routine dewaxing; With the HCL room temperature effect of 0.2mmol/L 3 minutes, the 1xPBS washing; 0.1% TritonX-100 room temperature 10 minutes, the 1xPBS washing; 50 μ g/ml Proteinase K room temperatures digestion 5 minutes, the 1xPBS washing; The gradient ethanol dehydration, drying at room temperature.
2, design, synthetic primer and digoxin labelled probe
According to the common HBV genotype B of China, C genom sequence, design primer and probe
RCA1?CATCCTCACAATA*C*C
RCA2?GGCTATTCTCCTC*C*C
RCA3?AGTATGGGAGTGG*G*C
RCA4?AGTTTGTCCAAGG*G*C
RCA5?ATGCAACTTTTTC*A*C
RCA6?CTAGCAGAGCTTG*G
RCA7?TACAAGAAGAACT*C
RCA8?CCCGGGACATATT*G
P59：5-GGCCACCTCTCTTTA-3
P83：5-DIGCGGCACAGCTTGGAGG-3
3, Plasmid safe ATP-dependent Dnase enzymic digestion:
On slide, drip the PSAD enzyme (reaction buffer 1 microlitre, ATP0.6 microlitre, PSAD enzyme 0.5 microlitre, H2O 13.3 microlitres) at present join organizationally, 37 ℃ 30 minutes.
4, rolling circle amplification:
Add RCA1, RCA2, RCA3, RCA4, RCA5, RCA6, RCA7, eight primers of RCA8 organizationally, each primer 0.8 μ l.95 ℃ of 3min, 55 ℃ of 15s, 33 ℃ of 15s, 20 ℃ of 10min; Again with above-mentioned eight primers: 0.8 each primer of μ l/, Phi29DNA polysaccharase 1 μ l, Phi29 buffer 1 μ l, BSA 0.2 μ l, d NTP 1.6 μ l, aseptic deionized water 4.2 μ l are added drop-wise to tissue.30 ℃ 18 hours.
5, HBVcccDNA in the original position pcr amplification hepatic tissue:
On hepatic tissue, drip original position PCR reaction solution (mark the primer 1 μ l of digoxin, Mg2+3 μ l, dNTP 5 μ l, Taq enzyme 1 μ l, PCR damping fluid 10 μ l), 95 ℃ 3 minutes, 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations, 72 ℃ 10 minutes.
6, signal detection:
With soaked in absolute ethyl alcohol 10 minutes, fixing 30 minutes of 4% Paraformaldehyde 96,1xTBS washing 3 times; 3%BSA room temperature 3 minutes, the anti digoxin antibody of dropping alkali phosphatase enzyme mark is hatched 2h for 37 ℃, the 1xTBS washing; Drip NBT/BCIP, dark place color development at room temperature 0.5h; 0.1% nuclear fast red is redyed karyon; Neutral gum mounting after the drying at room temperature, the microscopically observation analysis also writes down the result.
Positive karyon is blueness, purple and hyacinthine, and negative karyon is red
Positive particle semi-quantitative analysis in the cell: (-) do not see positive findings; Exist on (+) nucleus be dispersed in, sparse positive particle; Positive particle is prone to see on (++) karyon; (+++) is between (++) and (++ ++); (++ ++) positive findings is covered with whole karyon.
Tissue slice is positive to be analyzed: according to the cell total amount of estimating in positive signal representation quantity/this sheet in the section, count 5-10 the visual field, calculate the MV of total positives expression rate in the liver cell.
7, specificity, sensitivity and repeatable experiment:
Use respectively behind the non-covalent closed hoop DNA of original position PCR (PCR-ISH), PSAD digestion HBV and stride breach original position PCR method (PSAD+RCA+PCR-ISH with RCA+ after the direct in-situ PCR method (PSAD+PCR-ISH) and the non-covalent closed hoop DNA of PSAD digestion HBV; The inventive method) carries out HBV cccDNA in situ detection, 10 increments in clinical source are originally carried out 3 times repeated experiments respectively.With hepatitis C patients, healthy subjects, transgenic mice hepatic tissue as negative control.
8, chb, hbv-liver cirrhosis, hepatocellular carcinoma patients with hepatic organize HBV cccDNA to detect:
Stride breach method of in situ PCR (PSAD+RCA+PCR-ISH) with RCA+ after the employing PSAD enzymic digestion and respectively 50 routine chbs, liver cirrhosis, hepatocellular carcinoma patient are carried out HBV cccDNA detection.
Three, experimental result
1, method specificity, sensitivity and stability experiment:
Be sample with chb, liver cirrhosis, liver cancer patient hepatic tissue respectively, divide three groups to carry out the specificity experiment:
I group: hepatic tissue direct in-situ PCR method (PCR-ISH);
The II group: hepatic tissue is directly striden breach original position PCR (PSAD+PCR-ISH) after PSAD digestion;
The III group: hepatic tissue is striden breach original position pcr amplification (PSAD+RCA+PCR-ISH, the inventive method) with RCA+ after PSAD digestion;
HBV cccDNA positive signal (Fig. 1～3) in the positive sample after all visible original position amplification is main with caryogram, is dense bluish voilet colour developing.Control group all negative (Fig. 4).Results suggest HBV-cccDNA mainly is present in the nucleus, is the hepatitis B virus persistent infection and the root of duplicating.
Figure 1A and Figure 1B adopt respectively PCR-ISH and PSAD+PCR-ISH method detect chronic hepatitis B patient hepatic tissue sample HBV cccDNA, and HBV cccDNA positive signal is weak (blueness of maying be seen indistinctly) very.
Fig. 1 C adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR (PSAD+RCA+PCR-ISH) method and detects chronic hepatitis B patient hepatic tissue sample HBV cccDNA, and HBV cccDNA positive signal (bluish voilet) mainly is distributed in liver cell nuclear (* 400).
Fig. 2 A and Fig. 2 B adopt respectively PCR-ISH and PSAD+PCR-ISH method detect hbv-liver cirrhosis patients with hepatic tissue sample HBV cccDNA, and HBV cccDNA positive signal is weak (blueness of maying be seen indistinctly) very.
Fig. 2 C adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR method and detects hbv-liver cirrhosis patients with hepatic tissue sample HBV-cccDNA, and HBV-cccDNA positive signal (bluish voilet) mainly is distributed in liver cell nuclear (* 400).
Fig. 3 A and Fig. 3 B adopt respectively PCR-ISH and PSAD+PCR-ISH method detect liver cancer patient hepatic tissue sample HBVcccDNA, and HBV cccDNA positive signal is weak (blueness of maying be seen indistinctly) very.
Fig. 3 C adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR (PSAD+RCA+PCR-ISH) method and detects liver cancer patient hepatic tissue sample HBV cccDNA, and HBV cccDNA positive signal (bluish voilet) mainly is distributed in liver cell nuclear (* 400).
Fig. 4 HCV adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR (PSAD+RCA+PCR-ISH) method and detects hepatitis C patients hepatic tissue sample HBV-cccDNA, and karyon is red, does not see HBV cccDNA positive signal (bluish voilet) (* 20);
Fig. 4 HCC adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR (PSAD+RCA+PCR-ISH) method and detects liver cancer patient hepatic tissue sample HBV cccDNA; Do not add primer in the reaction system; Karyon is red, does not see HBVcccDNA positive signal (bluish voilet) (* 20);
Fig. 4 health adult adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR (PSAD+RCA+PCR-ISH) method and detects healthy subjects hepatic tissue sample HBV cccDNA, and karyon is red, does not see HBV cccDNA positive signal (bluish voilet) (* 20);
Fig. 4 transgenic mice adopts PSAD digestion+RCA+ of the present invention to stride breach original position PCR (PSAD+RCA+PCR-ISH) method and detects transgenic mouse hepatic tissue sample HBV cccDNA, and karyon is red, does not see HBV cccDNA positive signal (bluish voilet) (* 20).
Adopt the inventive method, 10 increments in clinical source are originally carried out duplicate detection 3 times, group difference does not have significance (p＜0.05) in the group.Show present method stability and better repeatable.
HBV cccDNA distributes and can know in the 50 routine patients with hepatic tissues through analyzing; HBV cccDNA is distributed in nucleus mostly in the hepatic tissue, is dense blueness or hyacinthine, is particulate state, arc or circular lumps; Mainly expressing (+) on nuclear and the nuclear membrane---do not wait between (++ ++).Negative patient and negative control liver cell karyon are red, do not see positive cell.
Four, experiment conclusion
The employing rolling circle amplification adds distribution and the location situation that the method for striding breach original position PCR detects HBVcccDNA in the hepatitis B patient liver cell, has specificity and safety preferably.
The characteristics of rolling circle amplification (RCA) are the closed hoop DNA that can only increase, thereby the HBV cccDNA that is used for increasing can improve the specificity of detection; Through the PSAD enzymic digestion, can remove the interference of non-covalent closed hoop DNA.Because the content of HBVcccDNA is lower usually, rolling circle amplification has the characteristic that the ability equality of temperature is rolled and duplicated in the presence of corresponding primer, on PSAD enzymic digestion basis, carries out rolling circle amplification, can improve the sensitivity of detection greatly; Add and stride breach PCR, further remove the PSAD enzyme and do not digest non-covalent completely closed hoop DNA, further strengthened specificity.
Confirm that to sum up present method has specificity, susceptibility and stability preferably.
Claims (9)
1. the detection method of direct viewing HBVcccDNA Distribution and localization in hepatic tissue, step is: the patients with hepatic tissue samples is fixed, and pcr amplification is carried out in digestion then, detects amplified production at last; It is characterized in that:
1) said sample fixedly is through 10% formaldehyde fixed, paraffin embedding, serial section with the hepatic tissue sample;
2) said digestion is to digest with proteolytic enzyme and PSAD enzyme;
3) said pcr amplification is on hepatic tissue section, to carry out original position to roll ring and add and stride the breach pcr amplification;
4) detection of amplified production is after rolling the anti digoxin antibody that ring adds dropping alkali phosphorus enzyme labelling on the hepatic tissue section after striding the breach pcr amplification, hatching certain hour, drips NBT/BCIP; Naked eyes direct viewing hepatic tissue section colour developing situation under the room temperature simple microscope, it is that HBVcccDNA expresses the positive that blueness, purple and hyacinthine particle person appear in nucleus, it is negative for HBVcccDNA expresses that red person appears in nucleus;
5) whole testing process is all carried out being loaded with on the glass slide of hepatic tissue section.
2. the described method of claim 1, said hepatic tissue sample fixed method is: with the hepatic tissue sample through 10% formaldehyde fixed, paraffin embedding, serial section, after the YLENE dewaxing, gradient ethanol dehydration, drying at room temperature.
3. the described method of claim 1, said digestion are to adopt the protease digestion hepatic tissue section to increase tissue permeability earlier, adopt non-covalent closed hoop DNA in the PSAD enzymic digestion hepatic tissue then.
4. the described method of claim 1 is carried out original position and is rolled ring and add that to stride the used instrument of breach pcr amplification be original position PCR appearance.
5. claim 1 or 4 described methods are carried out original position and are rolled ring and add that to stride the breach pcr amplification be on tissue slice, to add the rolling circle amplification primer and stride the breach primer, roll ring and add and stride breach original position pcr amplification HBVcccDNA.
6. the described method of claim 1, the detection of said amplified production are the anti digoxin antibodies that on tissue slice, drips alkali phosphatase enzyme mark, hatch 1 * TBS washing for 37 ℃; Drip NBT/BCIP, the dark place color development at room temperature; 0.1% nuclear fast red is redyed karyon, and neutral gum mounting after the drying at room temperature, microscopically are observed and the record result.
7. but distribution and the positioned detection test kit of a direct viewing HBVcccDNA in hepatic tissue; Contain stain remover, dNTP and 1 * PBS, it is characterized in that also containing following one or more materials: tissue slice fixing agent, deparaffinization reagents, closed reagent, proteolytic enzyme, PSAD enzyme, Phi29 archaeal dna polymerase, roll ring primer and digoxigenin labeled primer.
8. the described test kit of claim 7 also contains anti digoxin antibody, 1 * TBS, NBT/BCIP, the nuclear fast red of Phi29 buffer, alkali phosphatase enzyme mark; And said tissue slice fixing agent is 10% formaldehyde; Said proteolytic enzyme is Proteinase K, and said deparaffinization reagents is a YLENE, and said closed reagent is that BSA, said stain remover are TritonX-100.
9. the described test kit of claim 7, its detection method is that the hepatic tissue sample is fixed, paraffin embedding digests, and carries out original position then and rolls ring and add and stride the breach pcr amplification, at last in microscopically naked eyes direct viewing hepatic tissue section color.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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