CN105821156A - Method for detecting or/and quantifying hepatitis B virus in vitro and primer and probe used by same - Google Patents
Method for detecting or/and quantifying hepatitis B virus in vitro and primer and probe used by same Download PDFInfo
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Abstract
The invention provides a primer, a primer pair and a probe for detecting and/or quantifying hepatitis B virus, wherein the sequence codes of the primer are SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4, the primer can form the primer pair, and the sequence codes of the probe are SEQ ID No. 5 or SEQ ID No. 6. The invention also provides a method for detecting or/and quantifying the hepatitis B virus in vitro. The method for detecting or/and quantifying the hepatitis B virus in vitro and the primer and the probe used by the method have high binding force with the hepatitis B virus and can accurately judge whether a sample contains the hepatitis B virus. The primer and/or the probe can quickly and accurately detect whether the sample contains the hepatitis B virus in vitro, and the content of the hepatitis B virus in the sample can be obtained by analyzing the detection result.
Description
Technical field
The present invention is related to a kind of method detecting virus in vitro, refers in particular to a kind of detection in vitro and/or the method for quantitative hepatitis B virus and the primer used, probe.
Background technology
Although hepatitis B vaccine is in Taiwan and the injection comprehensively of many countries, hepatitis B (chronichepatitisB) is still one of modal infectious disease in the whole world, according to statistics, still there are 300,015,000 artificial hepatitis B chronic infection in the whole world, and wherein more than 75% in the Asian-Pacific area, estimated in Taiwan infect chronic hepatitis B close to four million peoples.Infect the patient of hepatitis B, in addition to causing the disease such as acute hepatitis, chronic hepatitis, calibration ordinary person has higher chance suffering from liver cirrhosis (livercirrhosis) or hepatocarcinoma (hepatocellularcarcinoma) simultaneously, and the whole world there are about 100,000,000 people every year and dies from hepatitis B relevant disease.For Taiwan, mortality of liver cancer is in the second of cancer related mortality rate, within 2011, has 8, and 022 people dies from hepatocarcinoma；Chronic hepatopathy and liver cirrhosis are then the 8th in the ten big causes of the death, within 2011, have 5, and 153 people die from liver related disease；In other words, because of the sum that hepatocarcinoma and hepatic disease are dead, account for all causes of death in Taiwan close to about one one-tenth.
Hepatitis B virus belongs to Hepadnaviridae Tobamovirus, for deoxyribose nucleic acid virus (DNAvirus), have 3,200 nucleotide, and hepatitis B virus at least ten kinds of genotype (A-J type), respectively the sequence differences of this genotype is more than 8%.The modal genotype of hepatitis B virus in the Asian-Pacific area is genotype B and genotype C virus, and Taiwan is particularly common with genotype B again.The hepatitis B virus of genotype B can further be subdivided into six hypotypes of B1-B6, and wherein, the hypotype such as B2-B5 is relatively conventional in Taiwan.Genotype of hepatitis B virus transfers the chance of chronic hepatitis and suffering from hepatic cancer to chance after being related to be infected by it is relevant, transfers chronic hepatitis to as being had higher chance by the hepatitis b virus infected patient of genotype A and genotype D；And had higher chance suffering from liver cirrhosis or hepatocarcinoma by the chronic hepatitis B patient of genotype C or genotype D.Recent studies have shown that relative to the chronic hepatitis B carriers of genotype B, have the chance of 2.35 times of suffering from hepatic cancer with the chronic hepatitis carrier of genotype C according to Taiwan.
In addition to upper described hepatitis b gene type can be as the instrument of prediction Patient suffering from hepatic cancer or liver cirrhosis, the viral DNA concentration in hepatitis B patient blood can be as prediction chronic hepatitis B patient suffering from hepatic cancer and the instrument of liver cirrhosis chance in the future.Specifically, previously having studied confirmation, no matter whether hepatitis B patient is that HbeAg is positive, whether has liver cirrhosis, liver function height, the DNA viruses amount in hepatitis B patient blood may be used to individually predict the chance of this patient suffering from hepatic cancer in the future.Separately having research to confirm, suffering from hepatic cancer and the patient being treated surgically, in its blood, hepatitis B DNA concentration is relevant to recurrence of PHC chance.In addition, although partly the HbsAg in chronic hepatitis B patient blood has been cleared by, but still can tested hemorrhage in containing hepatitis B virus DNA, therefore, when using chemotherapeutic agents (chemotherapyagents) or immunosuppressant (immunosuppressiveagents) to treat in the future, hepatitis B occurs that in these patients the chance of reactivation is the biggest, the most still should periodically follow the trail of hepatitis B virus DNA concentration in its blood, in order to predict whether hepatitis B virus is re-activated.
From the foregoing, it will be observed that by hepatitis b virus infected patient except detecting its genotype of hepatitis B virus, and should periodically follow the trail of presence or absence and the concentration thereof of internal hepatitis B virus, prevention and the effect for the treatment of could be reached for liver related disease.Although suggestion to carry out drawing blood and ultrasound investigation for the every 3-6 of chronic hepatitis B patient month the most clinically, to follow the trail of acute attack or liver cirrhosis, the generation of hepatocarcinoma whether having hepatitis B.But, chronic hepatitis B genotype and hepatitis B virus DNA are not listed in by the conventional tracking project taken a blood sample at present at blood level, it main reason is that, the detection method used clinically at present has the disappearance that testing cost is high, detect overlong time, such as instant inverse transcription polymerase chain reaction (RT-PCR) needs at least two primer and probe to begin to overcome hepatitis B sequence heterogeneity, otherwise its accuracy is the highest, and at least to spend the testing cost of New Taiwan Currency 1600 yuan.And with fully-automated synthesis instrument (Ampliprepreal-timePCRsystem, RocheMolecularSystems, Branchburg, NJ) analyze the quantitative expense of virus in blood DNA about must New Taiwan Currency 2000 yuan, and the time of detecting must just can complete in about 2-4 week.
It follows that current existing detection method cannot be commonly applied to detection follows the trail of chronic hepatitis-B infection patient, reach prevention or follow the trail of effect that related liver disease occurs.Therefore, detection and/or the important topic that method is current medical research of quantitative hepatitis B virus of a kind of novelty are developed.
Summary of the invention
Present invention is primarily targeted at a kind of probe detecting hepatitis B virus of offer, its sequential coding is SEQIDNo.5 or SEQIDNo.6, and it has high-bond with hepatitis B virus, and can differentiate in sample whether contain hepatitis B virus exactly.In general, this probe is able to synthetic mode or is prepared from the aggregated polymerase chain reaction of specific primers designed.
Another object of the present invention is to provide a kind of quantitative in vitro and/or method of detection hepatitis B virus, it can obtain testing result rapidly and accurately, and by the comparison with standard curve, learn the content of hepatitis B virus in sample to be tested, and testing cost can be reduced.
Third object of the present invention is to provide a kind of primer detecting hepatitis B virus, it has high specificity for hepatitis B virus, thereby it is amplified the fragment of hepatitis B virus in sample to be tested, and can interpolate that and whether sample to be tested contains hepatitis B virus and its content.
For above-mentioned purpose can be reached, taken off a kind of for detection and/or the quantitatively primer of hepatitis B virus or primer pair, wherein, the sequential coding of this primer is SEQIDNo:1, SEQIDNo:2, SEQIDNo:3 or SEQIDNo:4, and this primer is to being made up of above-mentioned primer, such as: this primer is to having the forward primer of coding SEQIDNo:1 and being encoded to the reverse primer of SEQIDNo:2, or encodes the forward primer of SEQIDNo:3 and be encoded to the reverse primer of SEQIDNo:4.
By above-mentioned primer or primer pair, another embodiment of the present invention discloses a kind of detection and/or the method for quantitative hepatitis B virus, its by the nucleic acid molecules of a sample to be tested and this primer or this primer to carrying out polymeric enzymatic amplification reaction, analyze this reaction result, it is judged that whether this sample to be tested contains hepatitis B virus and content thereof.
It is preferred that this step b further includes offer a certain amount of molecule, for example, this quantitative molecular be a fluorescent material or one have electroactive DNA be fitted together to agent.
And according to the technical field of the invention and have the general knowledge of usual skill, the mode analyzing polymeric enzymatic amplification reaction is numerous, such as with colloid electrophoresis carry out DNA fragmentation analysis, with the signal intensity of optical instrument analysis of fluorescence molecule, analyze DNA with electrochemical detection and be fitted together to the signal etc. of agent.
Embodiments of the invention provide a kind of for detecting and/or the probe of quantitative hepatitis B, and its sequential coding is SEQIDNo:5 or SEQIDNo:6.
Another embodiment of the present invention is disclosed in the method for vitro detection hepatitis B virus, and it comprises the steps of
Step a: take the nucleic acid molecules of a sample to be tested；
Step b: above-mentioned probe is provided；
Step c: the nucleic acid molecules of this sample to be tested and this probe are carried out heterozygosis reaction；
Step d: measure this probe respectively and carry out the binding ability before and after heterozygosis reaction with this sample to be tested, analyze according to this in this sample to be tested and whether contain hepatitis B virus, wherein, when this probe and this sample to be tested carry out the reacted binding ability of heterozygosis carry out the binding ability before heterozygosis reaction more than it time, represent that this sample to be tested contains hepatitis B virus.
It is preferred that this sample to be tested is blood, serum, blood plasma or body fluid.
Preferably, this method detecting hepatitis B virus in vitro further includes step e, after being located at this step d, one master sample containing variable concentrations hepatitis B virus is provided, heterozygosis reaction is carried out respectively with this probe, and measure respectively this master sample and the binding ability of this probe, the binding ability preparation corresponding thereto of hepatitis B virus concentration is become a standard curve, and by the binding ability of this sample to be tested compared with this standard curve, obtain the concentration of hepatitis B virus DNA in this sample to be tested.
It is preferred that the measurement standard of binding ability is fluorescence intensity, color change, resistance variations or optical signalling in step d, the operation with resistance variations is the easiest the most again.
The invention have the benefit that
The present invention provides a kind of detection in vitro and/or the method for quantitative hepatitis B virus and the primer used, probe, and it has high-bond with hepatitis B virus, it is possible to differentiate in sample whether contain hepatitis B virus exactly.Accordingly, primer of the present invention and/or probe can detect in sample whether contain hepatitis B virus the most in vitro, and known the content of hepatitis B virus in this sample by analyzing testing result.
Accompanying drawing explanation
Fig. 1 be the first primer of the present invention to, the second primer to and JX-1-1 primer the analysis result of polymerase chain reaction is carried out for different corpse or other object for laboratory examination and chemical testing.
Fig. 2 is the result that detection the first test strip and a corpse or other object for laboratory examination and chemical testing react before and after's resistance value.
Fig. 3 is the result that detection the second test strip and a corpse or other object for laboratory examination and chemical testing react before and after's resistance value.
Fig. 4 is the result that detection the 3rd test strip and a corpse or other object for laboratory examination and chemical testing react before and after's resistance value.
Fig. 5 is detection the first test strip and the result reacting before and after's resistance value containing the corpse or other object for laboratory examination and chemical testing that hepatitis B virus concentration is 1ng/ μ L.
Fig. 6 is detection the first test strip and the result reacting before and after's resistance value containing the corpse or other object for laboratory examination and chemical testing that hepatitis B virus concentration is 100pg/ μ L.
Fig. 7 is detection the first test strip and the result reacting before and after's resistance value containing the corpse or other object for laboratory examination and chemical testing that hepatitis B virus concentration is 10pg/ μ L.
Detailed description of the invention
Unless otherwise defined, technology that description and the claim in the present invention is used and the meaning of scientific term, its with the technical field of the invention and have usual skill be commonly understood by identical.If contradictory situation, it is as the criterion with present invention.
The present invention is taken off for detection and/or quantitatively hepatitis B virus primer can be in conjunction with the fragment of hepatitis B virus DNA, and its sequential coding is SEQIDNo:1, SEQIDNo:2, SEQIDNo:3 or SEQIDNo:4.
The present invention taken off for detection and/or quantitatively hepatitis B virus primer to can in conjunction with and amplify the DNA fragmentation of hepatitis B virus, wherein, the sequential coding of the forward primer of this primer pair is SEQIDNo:1 or SEQIDNo:3, further, the sequential coding of its reverse primer is SEQIDNo:2 or SEQIDNo:4.
The present invention is taken off for detection and/or the probe of quantitative hepatitis B, and its sequential coding is SEQIDNo:5 and SEQIDNo:6, has the function that the DNA fragmentation with hepatitis B virus is combined.
The taken off primer of the present invention and primer pair, designed by the whole genome sequence of comparison different shaped hepatitis B virus and obtain.
The taken off probe of the present invention, the primer formed by SEQIDNo:1 and SEQIDNo:2 with sequential coding to or the primer pair that formed by SEQIDNo:3 and SEQIDNo:4 of sequential coding, amplified the DNA fragmentation of sequential coding SEQIDNo:5 or SEQIDNo:6 from hepatitis B virus DNA by polymerase chain reaction.
And the taken off primer of the present invention, primer to or probe be also able to synthetic mode and obtained.So-called " synthetic mode " word refers to that being sequentially formed by connecting by aminoacid by manual type is a polypeptide, comprises chemical synthesis and Peptide synthesizer.In general, chemical synthesis can be divided into solid phase polypeptide synthesis and Liquid phase peptides synthesis method, and wherein, Liquid phase peptides synthesis method must carry out extracting operation after completing each amino acid whose connection；Subsequent amino-acid first by the N terminal amino acid covalently bonded of be intended to polypeptide to polymer beads, is sequentially connected by the way of specificity bond, is finally synthesizing this polypeptide by solid phase polypeptide synthesis the most again.Compared to Liquid phase peptides synthesis method, solid phase polypeptide synthesis need not purification of intermediates, has preferable productivity, and can significantly shorten the response time, the most relatively has advantage, therefore, by the polypeptide synthesis method that the most widely people is used at present in the synthesis of long-chain polypeptide.
Such as the technical field of the invention and have well known to usual skill, specific primers designed by the present invention or primer are to being combined with the nucleic acid molecule complementation of hepatitis B virus, therefore, by analyzing the result of polymerase chain reaction, can detect that and whether sample to be tested contains hepatitis B virus and content thereof.
Hereinafter, for illustrating further many effects of the present invention, will elaborate for embodiment respectively, these embodiments are the example explained orally, and any vocabulary used in it is not limiting as description of the invention and the scope of claims and meaning.
Embodiment one: design primer pair
According to the information bank of country fossil data center (NationalCenterforBiotechnologyInformation, NCBI), learn all types of hepatitis B virus whole genome sequence.After analyzing via comparison, design one first primer pair, comprise the sequence being encoded to SEQIDNo.1 and SEQIDNo.2, and one second primer pair, comprise the sequence being encoded to SEQIDNo.3 and SEQIDNo.4.
Embodiment two: prepare probe
Respectively with this first primer designed in embodiment one to and this second primer to carrying out polymerase chain reaction, 35 circulations, in hbv nucleic acid molecule, obtain specific DNA fragmentation, and, will respectively this fragment with nucleic acid sequencing set group (And automatic nucleic acid genetic analysis systems (automatedABI Terminatorv3.1CycleSequencingKit)3100, Biosystems, CA, USA) verify, learn that the sequence of respectively this fragment is respectively SEQIDNo.5 and SEQIDNo.6, and will be respectively as the first probe and the second probe, for subsequent embodiment.
Embodiment three: purification hepatitis B virus
Take hepatitis B blood sample, it is centrifuged processing with centrifuge, collect the leukocytic cream (buffycoat) 1 milliliter of each blood sample, add 4 milliliters of erythrocyte cracked liquids (RBCLysisBuffer), uniformly mix, put 10 minutes at room temperature, it is centrifuged 5 minutes with rotating speed 3000rmp again, after removing its supernatant, adds the erythrocyte cracked liquid of 1 milliliter, and precipitate (pellet) is broken up, is again centrifuged 2 minutes.After removing supernatant, then plasmasome is resuspended in the erythrocyte cracked liquid of 3 milliliters.Put 30 minutes at 37 DEG C, add the RNaseA (10mg/mL) (Amresco, OH, USA) of 6 μ L, and carry out shaking 30 minutes with the rotating speed of 150rmp at 37 DEG C.Add the Protein Precipitation Solution (proteinprecipitationsolution, RBCBioscience) of about 1 milliliter, shake, be more at room temperature centrifuged 5 minutes with 3000rmp, it is thus achieved that a supernatant.The isopropyl acetone of about 3 milliliters is added in this supernatant, uniformly mixes, make DNA separate out.After being centrifuged 5 minutes with 3000rmp, remove supernatant completely, take out DNA, again the ethanol of 1 milliliter, concentration 70% is uniformly mixed with this DNA, it is centrifuged 5 minutes with 3000rmp, removes its supernatant, treat that ethanol volatilizees, add DNA aqua liquid and carry out back dissolving, complete the purification of hepatitis B blood sample.
With fully-automated synthesis instrument (Ampliprepreal-timePCRsystem, RocheMolecularSystems, Branchburg, NJ) measure the concentration of hepatitis B virus DNA in this blood sample.
Embodiment four: the hepatitis B virus of detection variable concentrations
Take a corpse or other object for laboratory examination and chemical testing for numbering 1-4, and, through knowable to the detection data of Taibei Pathology Core, the hepatitis B virus concentration contained in a corpse or other object for laboratory examination and chemical testing for numbering 1 to 4 is sequentially 5.53X105copies/mL、2.71X104copies/mL、1.21X107copies/mL、2.61X106copies/mL.By respectively this corpse or other object for laboratory examination and chemical testing respectively with this first primer to, this second primer to and JX-1-1 primer to carrying out polymerase chain reaction, result is as shown in Figure 1.Wherein, JX-1-1 primer, to for having delivered the primer pair being used in detection hepatitis B in document, has forward primer that sequential coding is SEQIDNo.7 and sequential coding is the reverse primer of SEQIDNo.8.And this polymerase chain reaction carried out is the technical field of the invention and has well known to usual skill, thus experiment flow not in this to go forth.
Shown by the testing result of Fig. 1, the present invention taken off the first primer to and this second primer content to being able to efficiently differentiate out variable concentrations hepatitis B virus.
Embodiment five: prepare test strip
Take this first probe, this second probe and Hx1-1 probe in the present embodiment to prepare respectively and become the first test strip, the second test strip and the 3rd test strip, wherein, Hx1-1 probe is disclosed in order to detect the probe of hepatitis B virus in prior document, and its sequential coding is SEQIDNo.9.
The flow process preparing test strip is as follows: respectively will dilute 100 times with phosphate buffer respectively by this probe, it is placed in the concussion of test tube oscillator uniformly, take 40 μ L again to drip in detection wafer surface, stand 1 hour at normal temperatures, make respectively this probe be binding on respectively on this detection wafer.Respectively this wafer will be cleaned with phosphate buffer again, in order to remove the probe not being attached on wafer, after dry respectively this wafer, complete to prepare respectively this test strip.
Embodiment six: test hepatitis B virus and the binding ability of probe
Take a corpse or other object for laboratory examination and chemical testing with hepatitis B virus, add phosphate buffer and dilute 100 times, be positioned over 95 DEG C of beaker water proofs and heat 10 minutes, and be positioned over rapidly 4 DEG C of cold preservations 90 seconds, after being cooled to 50 DEG C in test tube concussion uniformly.Take this first test strip, this second test strip and the 3rd test strip, survey its resistance value the most respectively, again this corpse or other object for laboratory examination and chemical testing solution 40 μ L is separately added into this first test strip, this second test strip and the surface of the 3rd test strip, 30 minutes are stood at 50 DEG C, after cleaning with phosphate buffer again, carrying out Electrochemical Detection, result is as shown in Figures 2 to 4.
Knowing that resistance value is the biggest when measured, represent that the number of probes that hepatitis B virus is combined in a corpse or other object for laboratory examination and chemical testing is more, the binding ability of display probe is stronger, on the contrary, when measured know that resistance value is less time, represent that hepatitis B virus is combined in a corpse or other object for laboratory examination and chemical testing number of probes is less, the binding ability of probe is poor.Further, the probe that binding ability is strong can detect more viral level, increases the accuracy of its detection according to this, and the situation reducing erroneous judgement diagnosis occurs.
According to described above, from the result of Fig. 2 to Fig. 4, this first test strip and this second test strip can actually in order to detect hepatitis B virus, and, this first test strip and this measured resistance value of the second test strip are apparently higher than the 3rd test strip.Thus result understands, and the present invention is taken off the first probe and the second probe the most relatively Hx1-1 probe has stronger binding ability, thus more viral level can be detected, makes the test piece comprising this first probe or this second probe can have good Detection accuracy.
Embodiment seven: the sensitivity of detection probe
Taking a hepatitis B corpse or other object for laboratory examination and chemical testing for numbering 3, its gDNA concentration is prepared as 1ng/ μ L, 100pg/ μ L and 10pg/ μ L, detects with this first test strip respectively, result is as shown in Figures 5 to 7.Thus result understands, even if the virus concentration in a hepatitis B corpse or other object for laboratory examination and chemical testing is 10pg/ μ L, this first test strip still can reach the purpose of detection.
The virus quantity that further can detect with Roche COBASAmpliPrep this first test strip of full-automatic nucleic acid extraction systematic analysis, finds that the viral load that this first test strip can detect is at least 2.85X107copies/mL.By the hepatitis B corpse or other object for laboratory examination and chemical testing of comparison numbering 3 at the actually measured viral load (1.21X10 of Pathology Core7Copies/mL), can more clearly show when the taken off probe of present invention hepatitis B virus concentration in the sample is 100pg/ μ L, still there is Detection results.
From the above results, the present invention is taken off, and probe has high sensitivity, and therefore, even if when sample contains a small amount of hepatitis B virus, the taken off probe of the present invention still has good recall rate, and can be combined with more hepatitis B virus.
More than it is only through respectively this embodiment and describes the present invention in detail, know this those skilled in the art without departing under spirit of the present invention, and any simple modification that the embodiment in description is done or change, all should be the claims in the present invention and contained.
Claims (9)
1. the primer being used for detection and/or quantitative hepatitis B virus, it is characterized in that, in order to combine the DNA of hepatitis B virus in sample, this primer choosing group that freely following primer is formed: sequence shown in sequence shown in sequence, SEQIDNo:3 shown in sequence shown in SEQIDNo:1, SEQIDNo:2 and SEQIDNo:4.
2. the primer pair being used for detection and/or quantitative hepatitis B virus, it is characterized in that, in order to combine the DNA of hepatitis B virus in sample, the forward primer choosing group that freely following forward primer is formed of this primer pair: forward primer shown in forward primer shown in SEQIDNo:1 and SEQIDNo:3；And its reverse primer choosing group that freely following reverse primer is formed: reverse primer shown in reverse primer shown in SEQIDNo:2 and SEQIDNo:4.
3. a detection and/or the method for quantitative hepatitis B virus, it is characterised in that comprise the steps of
A () takes the nucleic acid molecules of a sample to be tested；
B () provides the hepatitis B virus DNA fragment in primer as claimed in claim 1 or primer pair, with this sample to be tested as claimed in claim 2 to carry out amplified reaction；
C () analyzes the result of this amplified reaction, learn whether have hepatitis B virus and content thereof in this sample to be tested according to this.
4. detect as claimed in claim 3 and/or the method for quantitative hepatitis B virus, it is characterised in that described step b further includes a certain amount of molecule of offer, and selects free fluorescent material and have the group that electroactive material is formed.
5. the probe being used for detection and/or quantitative hepatitis B, it is characterised in that sequence selects the group that free one sequence is formed: SEQIDNo:5 and SEQIDNo:6.
6. the method detecting hepatitis B virus in vitro, it is characterised in that include the following step:
A () takes the nucleic acid molecules of a sample to be tested；
B () provides probe as claimed in claim 5；
C the nucleic acid molecules of this sample to be tested and this probe are carried out heterozygosis reaction by ()；
D () is measured this probe respectively and is carried out the binding ability before and after heterozygosis reaction with this sample to be tested, analyze according to this in this sample to be tested and whether contain hepatitis B virus, wherein, when this probe and this sample to be tested carry out the reacted binding ability of heterozygosis carry out the binding ability before heterozygosis reaction more than it time, represent that this sample to be tested contains hepatitis B virus.
7. the method detecting hepatitis B in vitro as claimed in claim 6, it is characterised in that described sample to be tested selects the group that free blood, serum, blood plasma and body fluid are formed.
8. the method detecting hepatitis B in vitro as claimed in claim 6, it is characterized in that, further include step e, after being located at described step d, one master sample containing variable concentrations hepatitis B virus is provided, heterozygosis reaction is carried out respectively with this probe, and measure respectively this master sample and the binding ability of this probe, the binding ability preparation corresponding thereto of hepatitis B virus concentration is become a standard curve, and by the binding ability of this sample to be tested compared with this standard curve, obtain the concentration of hepatitis B virus DNA in this sample to be tested.
9. the method detecting hepatitis B in vitro as claimed in claim 6, it is characterised in that in step d, the measurement standard of binding ability is by the group selecting the change of free fluorescence intensity, color, resistance variations and optical signalling to be formed.
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| TW103138948 | 2014-11-10 | ||
| TW103138948A TWI586809B (en) | 2014-11-10 | 2014-11-10 | In vitro detection and / or quantification of hepatitis B virus and its use of the primer, probe |
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| Publication Number | Publication Date |
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| CN105821156A true CN105821156A (en) | 2016-08-03 |
| CN105821156B CN105821156B (en) | 2019-09-27 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0569237A2 (en) * | 1992-05-06 | 1993-11-10 | Gen-Probe Incorporated | Nucleic acid amplification oligonucletodies and probes to human hepatitis B virus |
| JPH11262399A (en) * | 1998-03-17 | 1999-09-28 | Srl Inc | Primer for detection of hepatitis b virus and detection of hepatitis b virus using the primer |
| WO2009122422A1 (en) * | 2008-03-31 | 2009-10-08 | Bigtec Private Limited | Probes and primers for detection of hepatitis b virus and a method thereof |
| CN102317474A (en) * | 2009-02-13 | 2012-01-11 | 彼格泰格私人有限公司 | Oligonucleotide probes and primers for detection of hepatitis b virus |
-
2014
- 2014-11-10 TW TW103138948A patent/TWI586809B/en active
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2015
- 2015-01-06 CN CN201510003745.8A patent/CN105821156B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0569237A2 (en) * | 1992-05-06 | 1993-11-10 | Gen-Probe Incorporated | Nucleic acid amplification oligonucletodies and probes to human hepatitis B virus |
| JPH11262399A (en) * | 1998-03-17 | 1999-09-28 | Srl Inc | Primer for detection of hepatitis b virus and detection of hepatitis b virus using the primer |
| WO2009122422A1 (en) * | 2008-03-31 | 2009-10-08 | Bigtec Private Limited | Probes and primers for detection of hepatitis b virus and a method thereof |
| CN102317474A (en) * | 2009-02-13 | 2012-01-11 | 彼格泰格私人有限公司 | Oligonucleotide probes and primers for detection of hepatitis b virus |
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| DRAMANE KANIA: "Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation", 《JOURNAL OF VIROLOGICAL METHODS 》 * |
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| Publication number | Publication date |
|---|---|
| CN105821156B (en) | 2019-09-27 |
| TW201617452A (en) | 2016-05-16 |
| TWI586809B (en) | 2017-06-11 |
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