CN105821156B - 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 method that the present invention detects virus about one kind in vitro refers in particular to a kind of detection in vitro and/or quantitative second
The method of Hepatitis virus and its used primer, probe.
Background technique
Although hepatitis B vaccine is in Taiwan and many comprehensive injections of country, hepatitis B (chronic
Hepatitis B) it is still one of most common infectious diseases in the whole world, according to statistics, still there are 300,005,000 Wan Renwei in the whole world
Hepatitis B chronic infection, and wherein more than 75% in the Asian-Pacific area, it is estimated to be in Taiwan chronic close to four million peoples infection
Hepatitis B.The patient for infecting hepatitis B, other than it will lead to the diseases such as oxyhepatitis, chronic hepatitis, while compared with normal person
There are higher chance suffering from liver cirrhosis (liver cirrhosis) or liver cancer (hepatocellular carcinoma), and it is complete
There are about 100,000,000 people to die of hepatitis B related disease every year in the world.For Taiwan, mortality of liver cancer is in cancer related mortality
The second of rate has 8,022 people to die of liver cancer for 2011；And chronic liver disease and cirrhosis are then the 8th in the ten big causes of the death,
There are 5,153 people to die of liver related disease within 2011；In other words, because of the sum of liver cancer and liver diseases death, Taiwan institute is accounted for
Have the cause of death close to one one-tenth or so.
Hepatitis type B virus belongs to Hepadnaviridae Tobamovirus, is that nuclifort is viral (DNA virus), tool
There are 3,200 nucleotide, and hepatitis type B virus at least ten kinds of genotype (A-J type), respectively the sequence differences of the genotype
Greater than 8%.The most common genotype of hepatitis B virus in the Asian-Pacific area is genotype B and genotype C virus, and Taiwan is again with gene
Type B is particularly common.The hepatitis type B virus of genotype B can further be subdivided into six hypotypes of B1-B6, wherein the Asias such as B2-B5
Type is relatively conventional in Taiwan.Genotype of hepatitis B virus is about switching to the chance of chronic hepatitis after being infected by it and suffer from liver
The chance of cancer is related, is such as switched to slowly by the hepatitis b virus infected patient of genotype A and genotype D chance with higher
Property hepatitis；And higher chance suffering from liver cirrhosis or liver cancer are had by the chronic hepatitis B patient of genotype C or genotype D.
According to Taiwan recent studies have shown that, relative to the chronic hepatitis B carriers of genotype B, the chronic hepatitis with genotype C
Carrier has the chance of 2.35 times of suffering from hepatic cancer.
In addition to the upper hepatitis b gene type can be used as the tool of prediction Patient suffering from hepatic cancer or cirrhosis, hepatitis B
Viral DNA concentration in blood samples of patients can be used as prediction chronic hepatitis B patient future suffering from hepatic cancer and cirrhosis chance
Tool.Specifically, previous research confirm, no matter whether hepatitis B patient is that HbeAg is positive, whether has cirrhosis, liver function
Can just, the DNA virus amount in hepatitis B patient blood can be used to individually predict the chance of patient suffering from hepatic cancer in future.Separately have
Studying confirms, suffering from hepatic cancer and the patient being treated surgically, hepatitis B DNA concentration and recurrence of PHC machine in blood
It can be related.In addition, although the HbsAg in part chronic hepatitis B patient blood has been cleared by, but can still be tested in bleeding
Containing hepatitis B virus DNA, therefore, used in future chemotherapeutic agents (chemotherapy agents) or immune
When inhibitor (immunosuppressive agents) is treated, there is the machine being re-activated in these patients in hepatitis B
Can be also larger, then hepatitis B virus DNA concentration in its blood still should be periodically tracked, to predict hepatitis type B virus whether again
Activation.
From the foregoing, it will be observed that its genotype of hepatitis B virus should be detected by being removed by hepatitis b virus infected patient, and answer
The presence or absence of internal hepatitis type B virus and its concentration are periodically tracked, the effect of prevention and treatment could be reached for liver related disease
Fruit.Although clinically suggesting carrying out blood drawing and ultrasonic examination in the every 3-6 of chronic hepatitis B patient months at present, it is with tracking
The no acute attack for having hepatitis B or the generation of cirrhosis, liver cancer.But in the conventional tracking project taken a blood sample at present
Chronic hepatitis B genotype and hepatitis B virus DNA are not included in blood level, main reason is that, it is clinical at present
Detection method used in upper has the missing that testing cost is high, detection time is too long, such as instant reverse transcription polymerase chain
Formula reaction (RT-PCR) needs at least two primer and probe beginning that can overcome hepatitis B sequence heterogeneity, and otherwise it is accurate
Property is not high, and at least to spend 1600 yuan of New Taiwan Currency of testing cost.And with full-automatic detecting instrument (
Ampliprep real-time PCR system, RocheMolecular Systems, Branchburg, NJ) analysis blood
Middle viral DNA quantifies expense about must be 2000 yuan of New Taiwan Currency, and detection time must could be completed for about 2-4 weeks.
It follows that existing detection method can not be commonly applied to detection tracking chronic hepatitis-B infection at present
Patient reaches prevention or tracks the effect of related liver disease occurs.Therefore, it develops the novel detection of one kind and/or quantifies
The method of hepatitis type B virus is the important topic of current medical research.
Summary of the invention
The main purpose of the present invention is to provide a kind of probe for detecting hepatitis type B virus, sequential coding is SEQ ID
No.5 or SEQ ID No.6 has high-bond with hepatitis type B virus, and can accurately differentiate in sample whether contain
Hepatitis type B virus.In general, which is able to artificial synthesized mode or with the aggregated enzyme chain of designed specific primers
Formula reaction is prepared.
Another object of the present invention is to provide method that is a kind of quantitative in vitro and/or detecting hepatitis type B virus,
Testing result can be rapidly and accurately obtained, and by the comparison with standard curve, learns hepatitis type B virus in sample to be tested
Content, and can reduce testing cost.
Third object of the present invention is to provide a kind of primer for detecting hepatitis type B virus, for hepatitis B
Poison has high specificity, obtains the segment of hepatitis type B virus in amplification sample to be tested whereby, and can judge sample to be tested
In whether contain hepatitis type B virus and its content.
For above-mentioned purpose can be reached, take off in embodiments of the present invention a kind of for detecting and/or quantifying hepatitis B
Poison primer or primer pair, wherein the sequential coding of the primer be SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 or
SEQ ID No:4, and the primer pair is made of above-mentioned primer, such as: the primer pair has the forward direction of coding SEQ ID No:1
Primer and the reverse primer for being encoded to SEQ ID No:2, or encode the forward primer of SEQ ID No:3 and be encoded to SEQ ID
The reverse primer of No:4.
By above-mentioned primer or primer pair, another embodiment of the present invention discloses a kind of detection and/or quantitative hepatitis B
The nucleic acid molecules of one sample to be tested are carried out polymeric enzymatic amplification with the primer or the primer pair and reacted by the method for poison, and analysis should
Reaction result judges whether contain hepatitis type B virus and its content in the sample to be tested.
A certain amount of molecule is provided preferably, further including in step b, for example, the quantitative molecular is a fluorescent material
Or one the electroactive DNA of tool be fitted into agent.
And the general knowledge of technical field and the usual skill of tool, analysis polymeric enzymatic amplification react according to the present invention
Mode is numerous, for example, with colloid electrophoresis carry out DNA fragmentation analysis, with the signal strength of optical instrument analysis of fluorescence molecule, with
Electrochemical detection analyzes the signal etc. that DNA is fitted into agent.
The embodiment of the present invention provides a kind of for detecting and/or quantifying the probe of hepatitis B, sequential coding SEQ
ID No:5 or SEQ ID No:6.
Another embodiment of the present invention is disclosed in the method for vitro detection hepatitis type B virus, and it includes the following steps:
Step a: the nucleic acid molecules of a sample to be tested are taken；
Step b: above-mentioned probe is provided；
Step c: the nucleic acid molecules of the sample to be tested are subjected to heterozygosis with the probe and are reacted；
Step d: the probe is measured respectively with the sample to be tested and carries out the binding ability that heterozygosis reacts front and back, analysis should accordingly
Whether contain hepatitis type B virus in sample to be tested, wherein when the probe and the sample to be tested carry out combination after heterozygosis is reacted
When ability is greater than its binding ability before carrying out heterozygosis reaction, indicate that the sample to be tested contains hepatitis type B virus.
Preferably, the sample to be tested is blood, serum, blood plasma or body fluid.
Preferably, the method for detecting hepatitis type B virus in vitro further includes a step e, it is located at after step d, mentions
For a master sample containing various concentration hepatitis type B virus, heterozygosis is carried out with the probe respectively and is reacted, and measures the respectively mark
The binding ability of hepatitis type B virus concentration corresponding thereto is prepared into a standard by the binding ability of quasi- sample and the probe
Curve, and the binding ability of the sample to be tested is compared with the standard curve, obtain hepatitis type B virus in the sample to be tested
The concentration of DNA.
Preferably, the measurement standard of binding ability is fluorescence intensity, color change, resistance variations or optics letter in step d
Number, wherein again the easiest with the operation of resistance variations.
The invention has the benefit that
The present invention provide it is a kind of detect in vitro and/or the method for quantitative hepatitis type B virus and its used primer,
Probe has high-bond with hepatitis type B virus, can accurately differentiate in sample whether contain hepatitis type B virus.According to
This, primer and/or probe of the present invention can quickly and accurately detect in vitro in sample whether contain hepatitis type B virus,
And it is able to know the content of hepatitis type B virus in the sample by analysis detection result.
Detailed description of the invention
Fig. 1 is that the first primer of the present invention polymerize a different specimen, the second primer pair and JX-1-1 primer
The analysis result of enzyme chain reaction.
Fig. 2 is the result for detecting the first test strip and reacting front and back resistance value with a specimen.
Fig. 3 is the result for detecting the second test strip and reacting front and back resistance value with a specimen.
Fig. 4 is the result for detecting third test strip and reacting front and back resistance value with a specimen.
Fig. 5 is to detect the first test strip to react front and back resistance with containing the specimen that hepatitis type B virus concentration is 1ng/ μ L
The result of value.
Fig. 6 is to detect the first test strip to react front and back electricity with containing the specimen that hepatitis type B virus concentration is 100pg/ μ L
The result of resistance value.
Fig. 7 is to detect the first test strip to react front and back electricity with containing the specimen that hepatitis type B virus concentration is 10pg/ μ L
The result of resistance value.
Specific embodiment
Unless otherwise defined, the meaning of the technology used in specification and claim of the invention and scientific term
Justice is identical as the general understanding of the technical field of the invention and the usual skill of tool.If contradictory situation, with the present invention
Subject to content.
The present invention takes off can be in conjunction with the piece of hepatitis B virus DNA for detecting and/or quantifying hepatitis type B virus primer
Section, sequential coding are SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 or SEQ ID No:4.
The present invention, which takes off, can combine for detecting and/or quantifying hepatitis type B virus primer pair and amplify hepatitis type B virus
DNA fragmentation, wherein the sequential coding of the forward primer of the primer pair be SEQ ID No:1 or SEQ ID No:3, also, its
The sequential coding of reverse primer is SEQ ID No:2 or SEQ ID No:4.
The present invention takes off for detecting and/or the probe of quantitative hepatitis B, sequential coding for SEQ ID No:5 and
SEQ ID No:6 has the function in conjunction with the DNA fragmentation of hepatitis type B virus.
The taken off primer of the present invention and primer pair, the whole genome sequence by comparing different shaped hepatitis type B virus design
?.
The taken off probe of the present invention is primer pair or sequence composed by SEQ ID No:1 and SEQ ID No:2 with sequential coding
Column are encoded to primer pair composed by SEQ ID No:3 and SEQ ID No:4, by polymerase chain reaction from hepatitis B
Malicious DNA cloning goes out the DNA fragmentation of sequential coding SEQ ID No:5 or SEQ ID No:6.
And the taken off primer of the present invention, primer pair or probe are also able to artificial synthesized mode and are obtained.So-called " artificial synthesized side
A formula " word, which refers to, to be sequentially formed by connecting amino acid by manual type as a polypeptide, includes chemical synthesis and Peptide systhesis
Instrument.In general, chemical synthesis can be divided into solid phase polypeptide synthesis and Liquid phase peptides synthesis method, wherein Liquid phase peptides synthesis
Method must carry out extracting operation after the connection for completing every monoamino-acid；Solid phase polypeptide synthesis is first by the N-terminal ammonia of be intended to polypeptide
In base acid covalently bonded to polymer beads, subsequent amino-acid is sequentially connected in such a way that specificity is bonded again thereafter,
It is finally synthesizing the polypeptide.Compared to Liquid phase peptides synthesis method, solid phase polypeptide synthesis does not need purification of intermediates, has preferable
Yield, and can substantially shorten the reaction time, the also relatively tool advantage in the synthesis of long-chain polypeptide, therefore, for more widely people at present
Used polypeptide synthesis method.
As well known to the technical field of the invention and the usual skill of tool, the specificity as designed by the present invention is drawn
Object or primer pair can be in conjunction with the nucleic acid molecule complementations of hepatitis type B virus, therefore, through the knot for analyzing polymerase chain reaction
Fruit can detect that in sample to be tested whether contain hepatitis type B virus and its content.
Hereinafter, will elaborate respectively for embodiment for more effects of the invention can be further explained, these implementations
Example is the example to explain, wherein used any vocabulary is not intended to limit the range of description of the invention and claims
And meaning.
Embodiment one: design primer pair
According to national fossil data center (National Center for Biotechnology Information,
NCBI data bank) learns all types of hepatitis type B virus whole genome sequences.After comparing analysis, one first is designed
Primer pair, sequence and one second primer pair comprising being encoded to SEQ ID No.1 and SEQ ID No.2, comprising being encoded to
The sequence of SEQ ID No.3 and SEQ ID No.4.
Embodiment two: probe is prepared
Polymerase chain reaction is carried out with the first primer pair and second primer pair designed in embodiment one respectively,
35 circulation, obtained from hbv nucleic acid molecule specificity DNA fragmentation, also, will respectively the segment with nucleic acid sequencing set
Group (Terminator v3.1Cycle Sequencing Kit) and automatic nucleic acid genetic analysis systems
(automated ABI3100, Biosystems, CA, USA) it is verified, learn the sequence difference of the respectively segment
For SEQ ID No.5 and SEQ ID No.6, and will be used respectively as the first probe and the second probe for subsequent embodiment.
Embodiment three: purifying hepatitis type B virus
Hepatitis B blood sample is taken, centrifugal treating is carried out with centrifuge, collects the leukocytic cream of each blood sample
4 milliliters of erythrocyte cracked liquids (RBC Lysis Buffer) is added in 1 milliliter of (buffy coat), uniformly mixes, is placed at room temperature
10 minutes, then centrifugation 5 minutes is carried out with revolving speed 3000rmp, after removing its supernatant, 1 milliliter of erythrocyte cracked liquid is added, and
And break up sediment (pellet), centrifugation 2 minutes is carried out again.After removing supernatant, then plasmasome suspended again
In 3 milliliters of erythrocyte cracked liquid.Put 30 minutes at 37 DEG C, be added 6 μ L RNase A (10mg/mL) (Amresco,
OH, USA), and concussion 30 minutes is carried out with the revolving speed of 150rmp at 37 DEG C.About 1 milliliter of Protein Precipitation Solution is added
(protein precipitation solution, RBC Bioscience), is shaken, then at room temperature with 3000rmp
Centrifugation 5 minutes is carried out, a supernatant is obtained.About 3 milliliters of isopropyl acetone is added in the supernatant, uniformly mixes, analyses DNA
Out.Be centrifuged after five minutes with 3000rmp, completely remove supernatant, take out DNA, then by 1 milliliter, the ethyl alcohol of concentration 70% with
The DNA is uniformly mixed, with 3000rmp carry out centrifugation 5 minutes, remove its supernatant, to ethyl alcohol volatilize, be added DNA aqua liquid into
Row back dissolving completes the purifying of hepatitis B blood sample.
With full-automatic detecting instrument (Ampliprep real-time PCR system,
RocheMolecular Systems, Branchburg, NJ) measure the concentration of hepatitis B virus DNA in the blood sample.
Example IV: the hepatitis type B virus of various concentration is detected
A specimen of number 1-4 is taken, also, the detection data through Taibei Pathology Core in a specimen for number 1 to 4 it is found that contain
Some hepatitis type B virus concentration is sequentially 5.53X105copies/mL、2.71X104copies/mL、1.21X107copies/
mL,2.61X106copies/mL.Will respectively the specimen respectively with the first primer to, second primer pair and JX-1-1 primer pair
Polymerase chain reaction is carried out, as a result as shown in Figure 1.Wherein, JX-1-1 primer pair is to have delivered to be used in detection in document
The primer pair of hepatitis B, with sequential coding be the forward primer of SEQ ID No.7 and sequential coding is SEQ ID No.8
Reverse primer.And the polymerase chain reaction that this is carried out is the technical field of the invention and has well known to usual skill,
Therefore not in this to go forth for experiment flow.
It is shown by the testing result of Fig. 1, taken off the first primer pair of the invention and second primer pair are able to effectively
Distinguish the content of various concentration hepatitis type B virus.
Embodiment five: test strip is prepared
First probe, second probe and Hx1-1 probe is taken to be prepared into the first detection examination respectively in the present embodiment
Piece, the second test strip and third test strip, wherein Hx1-1 probe is disclosed in prior document to detect B-mode liver
The probe of scorching virus, sequential coding are SEQ ID No.9.
The process for preparing test strip is as follows: respectively will dilute 100 times respectively with phosphate buffer by the probe, and be placed in examination
Pipe oscillator concussion uniformly, then take 40 μ L drops in detection wafer surface, at normal temperature standing 1 hour, make respectively the probe be bonded to
Respectively on the detection chip.The respectively chip will be cleaned with phosphate buffer again, to remove the probe not being attached on chip, done
After dry respectively chip, preparation respectively test strip is completed.
Embodiment six: the binding ability of test hepatitis type B virus and probe
Take one with hepatitis type B virus a specimen, be added phosphate buffer dilute 100 times, be placed in 95 DEG C of beakers every
Water heats 10 minutes, and is placed in 4 DEG C rapidly and refrigerates 90 seconds, uniform in test tube concussion after being cooled to 50 DEG C.Take first detection
Test piece, second test strip and the third test strip first survey its resistance value respectively, then 40 μ L of the specimen solution are added respectively
The surface for entering first test strip, second test strip and the third test strip stands 30 minutes at 50 DEG C, then with
After phosphate buffer cleaning, Electrochemical Detection is carried out, as a result as shown in Figures 2 to 4.
Know that resistance value is bigger when measured, indicates that the number of probes in conjunction with hepatitis type B virus in a specimen is more, display
The binding ability of probe is stronger, on the contrary, when it is measured know that resistance value is smaller when, indicate in conjunction with hepatitis type B virus in a specimen
Number of probes it is less, the binding ability of probe is poor.Also, the strong probe of binding ability can detect more viral level,
Increase the accuracy of its detection accordingly, the situation for reducing erroneous judgement diagnosis occurs.
According to above description, by the result of Fig. 2 to Fig. 4 it is found that first test strip and second test strip are certain
It can be to detect hepatitis type B virus, also, first test strip and the measured resistance value of the second test strip are bright
It is aobvious to be higher than third test strip.Thus result is it is found that the present invention takes off the first probe and the second probe respectively compared with Hx1-1 probe tool
There is stronger binding ability, thus can detect more viral level, makes the examination comprising first probe or second probe
Piece can have good Detection accuracy.
Embodiment seven: the sensitivity of detection probe
Its gDNA concentration is prepared as 1ng/ μ L, 100pg/ μ L and 10pg/ μ L by the hepatitis B specimen for taking number 3, respectively
It is detected with first test strip, as a result as shown in Figures 5 to 7.Thus result is it is found that even if in a hepatitis B specimen
Virus concentration be 10pg/ μ L when, which still can achieve the purpose of detection.
Further with the full-automatic nucleic acid extraction network analysis of Roche COBAS AmpliPrep the first test strip energy
The virus quantity of detection, the it is found that viral load for first test strip can detect are at least 2.85X107copies/mL.By it
Compare a hepatitis B specimen for number 3 the viral load (1.21X10 actually measured in Pathology Core7It copies/mL), can be brighter
When really showing the taken off probe of the present invention hepatitis type B virus concentration being 100pg/ μ L in the sample, still with detection effect.
From the above results, the taken off probe of the present invention has high sensitivity, therefore, even if containing a small amount of second in sample
When Hepatitis virus, the present invention is taken off, and probe still has good recall rate, and can be with more hepatitis type B virus knot
It closes.
The above is only by the way that respectively the present invention will be described in detail for the embodiment, the known those skilled in the art is not departing from essence of the invention
Under mind, and for any simple modification or variation that the embodiment in specification is made, the claims in the present invention institute should be
Cover.
Claims (6)
1. a kind of for detecting and/or quantifying the primer of hepatitis type B virus, which is characterized in that combine B-mode liver in sample
The DNA of scorching virus, the primer are selected from the group as composed by following primer: sequence shown in SEQ ID No:1, SEQ ID No:2 institute
Show sequence shown in sequence shown in sequence, SEQ ID No:3 and SEQ ID No:4.
2. a kind of for detecting and/or quantifying the primer pair of hepatitis type B virus, which is characterized in that B-mode in sample to combine
The forward primer of the DNA of hepatitis virus, the primer pair are selected from the group as composed by following forward primer: shown in SEQ ID No:1
Forward primer shown in forward primer and SEQ ID No:3；And its reverse primer is selected from the group as composed by following reverse primer:
Reverse primer shown in reverse primer shown in SEQ ID No:2 and SEQ ID No:4.
3. a kind of for detecting and/or quantifying the probe of hepatitis B, which is characterized in that sequence is selected from and is made of following sequence
Group: SEQ ID No:5 and SEQ ID No:6.
4. a kind of method for preparing hepatitis type B virus test strip, which is characterized in that comprise the steps of
(a) primer, primer pair as claimed in claim 2 or probe as claimed in claim 3 as described in claim 1 are provided；
(b) probe is diluted with phosphate buffer, and be uniformly mixed；
(c) it takes the mixing drop of step (b) to detect wafer surface in one, is bonded to the probe on the detection chip；
(d) probe not being attached on the detection chip is removed；
(e) dry detection chip, obtains a hepatitis type B virus test strip.
5. the method for preparation hepatitis type B virus test strip as claimed in claim 4, which is characterized in that in step (b), the spy
Needle dilutes 100 times with phosphate buffer.
6. the method for preparation hepatitis type B virus test strip as claimed in claim 4, which is characterized in that in step (c), take step
Suddenly the 40 μ L drop of mixed liquor of (b) is in the detection wafer surface.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
|---|---|
| CN105821156A CN105821156A (en) | 2016-08-03 |
| CN105821156B true CN105821156B (en) | 2019-09-27 |
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|---|---|---|---|
| CN201510003745.8A Expired - Fee Related CN105821156B (en) | 2014-11-10 | 2015-01-06 | Method for detecting or/and quantifying hepatitis B virus in vitro and primer and probe used by same |
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| CN (1) | CN105821156B (en) |
| TW (1) | TWI586809B (en) |
Citations (2)
| 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 |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2014
- 2014-11-10 TW TW103138948A patent/TWI586809B/en active
-
2015
- 2015-01-06 CN CN201510003745.8A patent/CN105821156B/en not_active Expired - Fee Related
Patent Citations (2)
| 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 |
| 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 |
Non-Patent Citations (5)
| Title |
|---|
| Detection of hepatitis B virus DNA by real-time PCR using TaqManMGB probe technology;Jin-Rong Zhao;《World J Gastroenterol 》;20050128;第11卷(第4期);508-510 * |
| PCR扩增HBV x基因特征序列检测乙肝病毒DNA;冯波等;《湘潭大学自然科学学报》;20110331;第33卷(第1期);85-88 * |
| Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation;Dramane Kania;《Journal of Virological Methods 》;20140218;第201卷(第2014期);24-30 * |
| Yan-Qin Lu等.Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.《World Journal of Gastroenterology》.2006,第12卷(第45期),7365-7370. * |
| 实时荧光定量PCR检测乙型肝炎病毒DNA的研究;袁媛;<中国优秀硕士学位论文全文数据库》;20071015(第4期);E060-38 * |
Also Published As
| Publication number | Publication date |
|---|---|
| TWI586809B (en) | 2017-06-11 |
| CN105821156A (en) | 2016-08-03 |
| TW201617452A (en) | 2016-05-16 |
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