CN107022543A - One kind extracts hbv nucleic acid with magnetic bead, extracts reagent, extracting method, quantitative detection kit - Google Patents
One kind extracts hbv nucleic acid with magnetic bead, extracts reagent, extracting method, quantitative detection kit Download PDFInfo
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Abstract
The present invention relates to gene by fluorescence quantitative detection technique field, and in particular to a kind of hbv nucleic acid extracts reagent, extracting method, quantitative detection kit.The magnetic bead is made up of three kinds of magnetic beads for being connected with target nucleotide sequences；Extracts reagent of the present invention includes the magnetic bead；Kit of the present invention uses extracts reagent of the invention.Magnetic bead of the present invention improves specificity and sensitivity that magnetic bead is extracted to hbv nucleic acid by connecting target nucleotide sequences in magnetic bead surfaces.The present invention extracts the extracting method of hbv nucleic acid, using the magnetic bead of the present invention, and in extraction process by way of heating up and cooling is combined, specificity and sensitivity that further lifting is extracted to hbv nucleic acid.Hepatitis type B virus of the present invention, which quantitatively detects, uses kit, employs the extracts reagent being made up of magnetic bead of the present invention, improves the specificity detected to hepatitis type B virus and sensitivity, hence it is evident that better than other domestic kits.
Description
Technical field
The present invention relates to gene by fluorescence quantitative detection technique field, and in particular to a kind of hbv nucleic acid extracts examination
Agent, extracting method, quantitative detection kit.
Background technology
Hepatitis type B virus (Hepatitis B Virus, HBV) can cause liver inflammatory lesion and multiple organ injury,
It is to seriously endanger one of epidemic virus of human health.After people's infection HBV, the non-purgee of viral persistence may develop into slowly
Property hepatitis, if without appropriate intervention, hepatic sclerosis, whole latter stage most occur at last for about 15%-40% Patients with Chronic HBV Infection
Hepatopathy and liver cancer, HBV infection person should actively carry out antiviral therapy, and monitor the change of its virus load.Therefore, hepatitis B
The malicious highly sensitive quantitative detection of nucleic acid is significant to the clinical conditions rational use of medicines and judging prognosis.
The genomic DNA of hepatitis type B virus is the structure of annulus double helix, is about 3.2kb.Two DNAs
Long, wherein 2/3 is double-spiral structure, 1/3 is single-stranded.Long-chain 5 ' end with 3 ' end without covalent attachment, but with a kind of protein
It is covalently attached to.5 ' ends of long-chain are combined with 250-300 to base complementrity.Long-chain is minus strand, and short chain is normal chain.The length of short chain is regarded
Virus and it is different, be typically about the 2/3 of 1.6-2.8kb, about long-chain.Space between short chain can be gathered by the DNA in virion
Synthase is filled.Hepatitis B is the minimum double-stranded DNA virus of the infection mankind being currently known.Thus HBV genome structure shows
Especially accurate concentration is obtained, its inhereditary material is made full use of.HBV can be divided into 8 genotype of A, B, C, D, E, F, G, H, and different
The pathogenic difference of genotype, the high Endemic Area of China's category HBV infection, the HBsAg positive rates of population are 9.09%.
Method at present for hepatitis type B virus quick detection is main by immunological method, method for gene chip and nucleic acid
Molecular biology method.Immunological detection method is mainly the antigen-antibody produced in detection infection course of hepatitis B virus and exempted from
Epidemic disease mark, most common method is EUSA (ELISA), is that specific antigen or antibody are adsorbed in into solid phase
On carrier, make itself and the corresponding antibodies in testing sample or antigen binding, then the antigen or antibody of enzyme-added mark, then add substrate
Colour developing, the content of determined antigen or antibody is calculated finally according to the color and luster depth.
Biochip technology is as probe, regularly and high density using a large amount of specific few core former times acid or genetic fragment
Ground is arranged, and is fixed on holder such as silicon chip, slide etc. of one piece of very little, then presses base with fluorescence labeling sample nucleic to be checked
Pair principle hybridize, it is scrubbed after by laser co-focusing fluorescing system detect hybridization signal intensities, then through computer analysis at
Reason data are so as to obtain the biological information of sample.Specific probe is fixed on slide, in PCR amplification hepatitis type B viruses point
Even if fluorescence is carried using the dNRP products of fluorescence labeling during type region, by the way that PCR primer and hepatitis type B virus chip is miscellaneous
There is fluorescence and determined in hybridization point after friendship at certain type.The detection of genetic mutation, Genotyping available for hepatitis type B virus.
Nucleic acid biology method is that DNA the or RNA fragments i.e. nucleic acid probe with label can be with complementary nucleotide sequence
Specific bond.The mark of nucleic acid probe can use nucleic and non-radioactive substance such as biotin, digoxin etc..Hepatitis type B virus
Nucleic acid probe have two kinds of DNA probe and rna probe.Nucleic acid probe method be mainly characterized by sensitiveness, specificity it is all stronger.
But ELISA high specificities, but ELISA detections HbsAg be negative sample may also hepatitis B virus DNA be
It is positive.Biochip technology has the advantages that automaticity is high, sensitivity is high, testing goal molecular weight is few, efficiency high, but
There is the defects such as cost height, the higher, poor repeatability of false positive rate.Therefore, such kit generally existing cost is high, false positive at present
Property rate is higher, poor repeatability, specificity sensitivity is low, the shortcomings of being unfavorable for automation.
In recent years, with the fast development of Protocols in Molecular Biology, technique of gene detection has been widely used in clinic,
This has benefited from the sensitivity of its height.DNA detections are important Medicine indication, especially antiviral therapy, and its confidence values will height
In other methods including immunization.Therefore, the gene diagnosis kit of relatively reliable stabilization is researched and developed to clinical guidance
Meaning seems more prominent.
DNA detection is mainly concerned with two aspects, the extraction of nucleic acid and the augmentation detection of nucleic acid.Viral nucleic acid in serum
Extraction, mainly have three kinds of methods, boiling method, post extraction method and magnetism separate method at present, and magnetic separation method has and reduces virus
Loss, quantitative accurate, easy to operate, efficiency high, the advantages of easily realizing automation, be widely used to the separation of nucleic acid with it is pure
Change.But, agents useful for same is extracted expensive, so as to limit its extensive use.
The method that clinically HBV-DNA is quantitatively detected is presently mainly to be based on Real-Time Fluorescent Quantitative PCR Technique, and it utilizes glimmering
Fluorescent Quantitative PCR amplification instrument, the dynamic change of fluorescence signal can be detected by fluorescence detection device, and reflection PCR's is each in real time
The level of amplification of circulation, while acquisition amplification curve is automatically analyzed by its own software carried, according to amplification curve and threshold
The intersection point (i.e. Ct values) and the shape of amplification curve of value, it can be determined that testing result.
Have a variety of HBV-DNA immue quantitative detection reagent boxes both at home and abroad at present, such kit is based on real time fluorescent quantitative
Round pcr, its extensive use in clinical detection protrudes and exposes many problems, and external kit purchase cost is high, sternly
Being widely applied for it is limited again；And domestic a variety of such kits show that sensitivity is not high, specificity is not strong, carries
The loss of process nucleic acid is taken seriously to wait some problems.In addition, the target molecule of pcr amplification product middle and high concentration can be with the shape of aerosol
Formula pollutes whole lab space, is required for strictly post-processing pcr amplification product, due to PCR high sensitivity, even
The template concentrations of 10 copies in reaction system can be detected, so as to bring serious false positive to experiment from now on.
Therefore, in the urgent need to the sensitivity and specificity of raising HBV-DNA immue quantitative detection reagent boxes.
The content of the invention
In order to overcome the defect of prior art, an object of the present invention is to provide a kind of high specific, high sensitivity and carried
Take hbv nucleic acid magnetic bead.
The second object of the present invention is to provide a kind of high characteristic, high sensitivity and extracts hbv nucleic acid extraction
Reagent.
The third object of the present invention is to provide the extraction that a kind of high specific, high sensitivity extract hbv nucleic acid
Method.
The fourth object of the present invention is to provide the reagent that a kind of high specific, high sensitivity quantitation detect hepatitis type B virus
Box.
In order to realize the above object the present invention is adopted the following technical scheme that：
One kind extracts hbv nucleic acid magnetic bead, by magnetic bead one, magnetic bead two and magnetic bead three according to 6： 6：1 ratio
Mixing composition；The magnetic bead one is the magnetic bead for being connected with target nucleotide sequences one, and magnetic bead two is to be connected with target nucleotide sequences two
Magnetic bead, magnetic bead three is to be connected with the magnetic beads of target nucleotide sequences three；
The target nucleotide sequences one are
5’-Biotin-AGACCACCAAATGCCCCTATCCTATCAACACTT-3’；
Target nucleotide sequences two are
5’-Biotin-GAGACCTTCGTCTGCGAGGCGAGGGAGTT-3’；
Target nucleotide sequences three are
5’-Biotin-C12-ATCGAGGGTAATCATTAACGGTGT-3’。
One kind extracts hbv nucleic acid extracts reagent, is made up of solution 1, solution 2, solution 3 and solution 4；
Wherein solution 1 by 0.1mol/L EDTA-Na2Solution, 2mol/L NaOH solution, 6mol/L guanidine hydrochloride it is molten
The tween solution that liquid, mass concentration are 20% is according to 8：8：40：21 volume ratio mixing composition；
Solution 2 by 4mol/L HCl solution and concentration for 2.5ug/ul magnetic bead solution according to 32：1.3 volume ratio is mixed
It is combined into；The magnetic bead is above-mentioned magnetic bead；
Solution 3 by 0.1mol/L EDTA-Na2Solution, 4mol/L NaCl solution, 1mol/L Tris-HCl bufferings
Liquid is with water according to 0.25：0.625：0.25：49 volume ratio mixing composition；
Solution 4 by solution 3 and mass concentration for 20% tween according to 20：1 volume ratio mixing composition.
A kind of extracting method for extracting hbv nucleic acid, including following operating procedure：1) sample to be tested is added
In centrifuge tube, the solution 1 in said extracted reagent is then taken to add in centrifuge tube, vibration is mixed, brief centrifugation；
2) take the solution 2 in said extracted reagent to add in centrifuge tube, after vibration is mixed, heated 10 minutes at a temperature of 50 DEG C
Afterwards, brief centrifugation, room temperature is cooled down 2 minutes；
3) by step 2) centrifuge tube after processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandon supernatant, add above-mentioned
Solution 4 in extracts reagent, vibration is mixed, brief centrifugation；
5) by step 3) centrifuge tube after processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandon supernatant, add above-mentioned
Solution 3 in extracts reagent, after standing 1 minute, abandons supernatant, that is, completes.
A kind of hepatitis type B virus, which quantitatively detects, uses kit, including said extracted reagent, PCR reaction solutions, negative Quality Control
Product, positive quality control product and standard items.
Optionally, the PCR reaction solutions are made up of the mixing of following solution：10 × PCR reactions buffer solution, 2mmol/L
DNTPs, mass concentration are mixed for 50% dimethyl sulphoxide solution, 4mol/L alkali solution of beet, 30pmol/ul target nucleotides primer
Close liquid, 30pmol/ul target nucleotides probe solution, 30pmol/ul interior label primers mixed liquor, 30pmol/ul internal standard probes molten
Liquid, 1U/ μ l hot resistant DNA polymerases, 1U/ μ l uracil dna glycosylases, water.
Optionally, the PCR reaction solutions are with buffer solution, 2mmol/L dNTPs, mass concentration by 10 × PCR reactions
50% dimethyl sulphoxide solution, 4mol/L alkali solution of beet, 30pmol/ul target nucleotides primer mixed liquor, 30pmol/ul targets
Nucleotide probe solution, 30pmol/ul interior label primers mixed liquor, 30 pmol/ul internal standard probes solution, 1U/ μ l heat-resistant dnas gather
Synthase, 1U/ μ l uracil dna glycosylases and water are according to 5：5：2：1：2.5：1：1：1：1.2：1：29.3 volume ratio mixing groups
Into.
Optionally, the dNTPs is made up of dNTP and dUTP.
Optionally, target nucleotide forward primer and target nucleus glycosides are contained in the 30pmol/ul target nucleotides primer mixed liquor
Sour reverse primer；Contain internal standard forward primer and internal standard reverse primer in 30pmol/ul interior label primer mixed liquors；
Its target nucleotide forward primer sequence is：
5’-AGACCACCAAATGCCCCTATCCTATCAACACTT-3’；
Target nucleotide reverse primer sequences are：
5’-GAGACCTTCGTCTGCGAGGCGAGGGAGTT-3’；
Internal standard forward primer sequence is：5’-ATCGAGGGTAATCATTAACGGTGT-3’；
Internal standard reverse primer sequences are：5’-CGTTCTCAAACGCCAGCGAAA-3’；
Target nucleotide probe sequence is：5 '-AGGCAGGTCCCCTAGAAGA-3 ', the end of target nucleotide probe 5 ' flag F AM
Fluorescent reporter group, 3 ' end mark MGB fluorescent quenching groups；
Internal standard probe sequence is：5 '-ACGGTGTGCCAGCATGTGTGGGA-3 ', internal standard probe 5` end mark HEX fluorescence
Reporter group, 3` ends mark BHQ1 fluorescent quenching groups.
Optionally, the negative quality-control product is the pUC57-T vector plasmids TMV-DNA without hepatitis B virogene
Fragment.
Optionally, the positive quality control product is 1.0x103copy/ml groups containing hepatitis B virogene target nucleotide sequences
PUC57-T vector plasmid fragments.
Optionally, the standard items are to contain 109 base-pair nucleosides at hepatitis B virus core gene terminal conserved sequences
The pUC57-T recombinant plasmids of acid fragment, the plasmid is bred in escherichia coli DH5a, specifically included：
Working standard 1：Target nucleotide sequences recombinant plasmid containing 1.0x107copy/ml；
Working standard 2：Target nucleotide sequences recombinant plasmid containing 1.0x106copy/ml；
Working standard 3：Target nucleotide sequences recombinant plasmid containing 1.0x105copy/ml；
Working standard 4：Target nucleotide sequences recombinant plasmid containing 1.0x104copy/ml.
Detect that the operating method of hepatitis type B virus concentration is in blood sample using mentioned reagent box：
(1) take blood sample to be measured to add in centrifuge tube, add internal standard compound；
(2) solution 1 in said extracted reagent is taken to be added in blood sample, vibration is mixed, brief centrifugation
(3) take the solution 2 in said extracted reagent to add in centrifuge tube, after vibration is mixed, 10 points are heated at a temperature of 50 DEG C
Zhong Hou, brief centrifugation, room temperature is cooled down 2 minutes；
(4) centrifuge tube after step (3) processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandoned in supernatant, addition
The solution 4 in extracts reagent is stated, vibration is mixed, brief centrifugation；
(5) centrifuge tube after step (4) processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandoned in supernatant, addition
The solution 3 in extracts reagent is stated, after standing 1 minute, supernatant is abandoned；
(6) magnetic bead on the centrifugation tube wall after also elution step (5) processing is reacted with PCR and is sucked in PCR pipe, together
When take negative quality-control product, positive quality control product, working standard is added in PCR pipe, covers lid；
(7) quantitative fluorescent PCR is carried out using quantitative real time PCR Instrument etc.：PCR pipe after step (6) processing is put into fluorescence
In the reactive tank of quantitative instrument, mark fluorescent radical species, sample ID and type are set, condition is carried out as shown in following table 1
Amplification：
Table 1
(8) interpretation of result：After quantitative fluorescent PCR reaction terminates, target curve is carried out in the curve and corresponding HBV to HBV
Analyze, it is possible to use the software that instrument is carried is analyzed, also can voluntarily be adjusted according to actual conditions respectively, record sample Ct values
Try to achieve the definite value result of each sample automatically with the standard curve drawn according to standard items；If sample amplification curve is S-type, there is Ct
Value and definite value result >=10IU/ml, can be judged to the positive；If sample amplification curve is straight, no Ct values are shown or without definite value knot
Fruit shows, can be judged to feminine gender.
Internal standard compound described in above-mentioned steps (1) is TMV DNA fragmentation, and its concentration is 1.0x104copy/ml。
The present invention extracts hbv nucleic acid magnetic bead, and target nucleotide sequences are connected in magnetic bead surfaces, improves magnetic bead
The specificity and sensitivity extracted to hbv nucleic acid.
The present invention extracts the extracting method of hbv nucleic acid, using the magnetic bead of the present invention, and in extraction process
In heat up and cooling combine by way of, further lifting to hbv nucleic acid extract specificity and sensitivity.
Hepatitis type B virus of the present invention, which quantitatively detects, uses kit, employs the extracts reagent being made up of magnetic bead of the present invention,
Improve the specificity detected to hepatitis type B virus and sensitivity, hence it is evident that better than other domestic kits；
Meanwhile, kit of the present invention uses the primer and probe of hepatitis B conserved sequence, with good specificity, should
The kit provided with the present invention, is capable of detecting when HBV eight genotype.
Further, kit of the present invention adds internal standard, can be with the presence of effective monitoring false negative.Add appropriate UNG
Enzyme can prevent the pollution of PCR primer.
Further, the magnetic bead and extracting method and rational PCR of target nucleotide sequences are connected with kit of the present invention
The preparation composition of reaction solution, height cooperation, improves sensitivity, the specificity of detection reagent each other, advantageously in certainly
Dynamicization, efficiency high remains ahead in such reagent at home at present.
Brief description of the drawings
Fig. 1 is the fluorescent quantitative PCR curve map of eight genotype standard items of HBV；
Fig. 2 is four HBV positive serums and hepatitis A virus, HCV, Epstein-Barr virus and cytomegalovirus
The fluorescent quantitative PCR curve map of known sample；
Fig. 3 is the fluorescent quantitative PCR curve map of 4 standard items；
Fig. 4 be 4 standard items concentration and Ct values between corresponding relation standard curve；
Fig. 5 is the fluorescent quantitative PCR curve map of 20 standard samples (including 1 negative internal reference product).
Embodiment
Technical scheme is described in detail below by specific embodiment.
Embodiment 1
The present embodiment provides a kind of extraction hbv nucleic acid magnetic bead, is pressed by magnetic bead one, magnetic bead two and magnetic bead three
According to 6：6：1 ratio mixing composition；The magnetic bead one is the magnetic bead for being connected with target nucleotide sequences one, and magnetic bead two is to be connected with target nucleus
The magnetic bead of nucleotide sequence two, magnetic bead three is the magnetic bead for being connected with target nucleotide sequences three；
Target nucleotide sequences one are
5’-Biotin-AGACCACCAAATGCCCCTATCCTATCAACACTT-3’；
Target nucleotide sequences two are
5’-Biotin-GAGACCTTCGTCTGCGAGGCGAGGGAGTT-3’；
Target nucleotide sequences three are
5’-Biotin-C12-ATCGAGGGTAATCATTAACGGTGT-3’。
Embodiment 2
The present embodiment provides a kind of extraction hbv nucleic acid extracts reagent, by solution 1, solution 2, the and of solution 3
Solution 4 is constituted；
Wherein solution 1 by 0.1mol/L EDTA-Na2Solution, 2mol/L NaOH solution, 6mol/L guanidine hydrochloride it is molten
The tween solution that liquid, mass concentration are 20% is according to 8：8：40：21 volume ratio mixing composition；
Solution 2 by 4mol/L HCl solution and concentration for 2.5ug/ul magnetic bead solution according to 32：1.3 volume ratio is mixed
It is combined into；The magnetic bead is the magnetic bead that embodiment 1 is provided；
Solution 3 by 0.1mol/L EDTA-Na2Solution, 4mol/L NaCl solution, 1mol/L Tris-HCl bufferings
Liquid is with water according to 0.25:0.625:0.25:49 volume ratio mixing composition；
Solution 4 by solution 3 and mass concentration for 20% tween according to 20：1 volume ratio mixing composition.
The composition of each component in the extracts reagent that this implementation is provided, referring specifically to table 2,
Table 2
The extracts reagent provided using the present embodiment extracts the extracting method of hbv nucleic acid, including following operation
Step：
1) sample to be tested is added in centrifuge tube, then takes the solution 1 in said extracted reagent to add in centrifuge tube, vibration
Mix, brief centrifugation；
2) take the solution 2 in said extracted reagent to add in centrifuge tube, after vibration is mixed, heated 10 minutes at a temperature of 50 DEG C
Afterwards, brief centrifugation, room temperature is cooled down 2 minutes；
3) by step 2) centrifuge tube after processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandon supernatant, add above-mentioned
Solution 4 in extracts reagent, vibration is mixed, brief centrifugation；
5) by step 3) centrifuge tube after processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandon supernatant, add above-mentioned
Solution 3 in extracts reagent, after standing 1 minute, abandons supernatant, that is, completes.
Embodiment 3
A kind of hepatitis type B virus of the present embodiment offer, which quantitatively detects, uses kit, includes the extraction examination that embodiment 2 is provided
Agent, PCR reaction solutions, negative quality-control product, positive quality control product and standard items；
PCR reaction solutions are 50% by 10 × PCR reactions buffer solution, 2mmol/L dNTPs, mass concentration by following solution
Dimethyl sulphoxide solution, 4mol/L alkali solution of beet, 30pmol/ul target nucleotides primer mixed liquor, 30pmol/ul target nucleus glycosides
Acid probe solution, 30pmol/ul interior label primers mixed liquor, 30 pmol/ul internal standard probes solution, 1U/ μ l hot resistant DNA polymerases,
1U/ μ l uracil dna glycosylases and water are according to 5：5：2：1：2.5：1：1：1：1.2：1：29.3 volume ratios mixing composition；
Reversely draw containing target nucleotide forward primer and target nucleotide wherein in 30pmol/ul target nucleotides primer mixed liquor
Thing；Contain internal standard forward primer and internal standard reverse primer in 30pmol/ul interior label primer mixed liquors；
Its target nucleotide forward primer sequence is：
5’-AGACCACCAAATGCCCCTATCCTATCAACACTT-3’；
Target nucleotide reverse primer sequences are：
5’-GAGACCTTCGTCTGCGAGGCGAGGGAGTT-3’；
Internal standard forward primer sequence is：5’-ATCGAGGGTAATCATTAACGGTGT-3’；
Internal standard reverse primer sequences are：5’-CGTTCTCAAACGCCAGCGAAA-3’；
Target nucleotide probe sequence is：5 '-AGGCAGGTCCCCTAGAAGA-3 ', the end of target nucleotide probe 5 ' flag F AM
Fluorescent reporter group, 3 ' end mark MGB fluorescent quenching groups；
Internal standard probe sequence is：5 '-ACGGTGTGCCAGCATGTGTGGGA-3 ', internal standard probe 5` end mark HEX fluorescence
Reporter group, 3` ends mark BHQ1 fluorescent quenching groups.
Negative quality-control product is the pUC57-T carriers without hepatitis B virogene group target nucleotide sequences；
Positive quality control product is 1.0x103The pUC57-T of copy/ml groups containing hepatitis B virogene target nucleotide sequences
Carrier；
Standard items are containing 109 base pair nucleotide fragments at hepatitis B virus core gene terminal conserved sequences
PUC57-T recombinant plasmids, the plasmid is bred in escherichia coli DH5a, is specifically included：
Working standard 1：Contain 1.0x107Copy/ml target nucleotide sequences recombinant plasmid；
Working standard 2：Contain 1.0x106Copy/ml target nucleotide sequences recombinant plasmid；
Working standard 3：Contain 1.0x105Copy/ml target nucleotide sequences recombinant plasmid；
Working standard 4：Contain 1.0x104Copy/ml target nucleotide sequences recombinant plasmid.
Wherein the concrete composition (50 μ l systems) of PCR reaction solutions, refers to table 3,
Table 3
Embodiment 4
The kit provided using embodiment 3 detects that to clinical blood sample and plasma sample serum sample is adopted
Diversity method is：One-shot injector extracts venous patient blood 1ml, as in the sterile test tube without anti-coagulants, in being stored at room temperature 2
Hour or 4 DEG C overnight after 1000g centrifuge 20 minutes, take supernatant.Serum should carry out next step operation immediately or be placed in -20 DEG C
Or -80 DEG C of preservations are to be checked.
The acquisition method of plasma sample is：One-shot injector extracts venous patient blood 1ml, is placed in sterile disposable anti-freezing
In test tube, centrifuged 15 minutes in 1000g in 30 minutes after collection of specimens, take supernatant.Supernatant should carry out immediately next step operation or
It is placed in -20 DEG C or -80 DEG C preservations to be checked.
Sample is deposited and movement requirement：It can be placed 72 hours at 2 DEG C -8 DEG C, or be placed in -20 DEG C and preserved 8 months；Sample is not
Easy multigelation, transport should be using foam box bag transport on the rocks.
The concrete operation step of detection method is：
(1) 2ml centrifuge tubes are taken and mark is carried out, take the clinical human serums of 320ul or plasma sample to add in centrifuge tube respectively,
1.5ul internal standard compounds are added in every part of sample, internal standard compound is TMV DNA fragmentation, and its concentration is 1.0x104copy/ml；
(2) take the solution 1 of 770ul extracts reagents to be added in centrifuge tube, concussion is mixed, brief centrifugation, open centrifuge tube
Lid；
(3) solution 2 of 160ul extracts reagents is taken to be added in centrifuge tube, concussion is mixed, 50 DEG C of heating 10min, instantaneously
Centrifugation, opens centrifugation lid, room temperature cooling 2min；
(4) collection magnetic bead 10min on magnetic frame is positioned over, supernatant is abandoned, the solution 4 of 1ml extracts reagents is added, concussion is mixed
Even, brief centrifugation opens centrifugation lid；
(5) collection magnetic bead 10min on magnetic frame is positioned over, supernatant is abandoned, the solution 3 of 1ml extracts reagents is added, stood
1min, abandons supernatant；
(6) centrifuge the magnetic bead on tube wall with the elution of 50ul PCR reaction solutions and sucked in PCR pipe, while negative Quality Control
Product, positive quality control product, working standard respectively takes 1ul to be separately added into PCR pipe, covers lid；
(7) quantitative fluorescent PCR：
The PCR pipe that step (6) is handled well is put into the anti-of quantitative real time PCR Instrument Q7 (ABI)/7500/step one plus
Answer in groove, mark fluorescent radical species, sample ID and type are set, expanded by the condition of following table 1：
Table 1
(8) interpretation of result：After quantitative fluorescent PCR reaction terminates, target curve is carried out in the curve and corresponding HBV to HBV
Analyze respectively, the software carried using instrument is analyzed, draw amplification curve, record sample Ct values and according to standard items Ct values
The standard curve drawn with concentration relationship tries to achieve the definite value result of each sample automatically；If sample amplification curve is S-type, there are Ct values
And definite value result >=10IU/ml, the positive can be judged to；If sample amplification curve is straight, no Ct values are shown or without definite value result
It has been shown that, can be judged to feminine gender；Testing result is needed to meet HBV negative controls simultaneously, and no CT values are shown；HBV internal standards are detected as sun
Property, and Ct≤40.HBV positive controls, detectable concentration value is between 1.58x102-1.58x103IU/ml.Four HBV qualitative references
Product：Equal test positive, and calibration curve coefficient correlation R2>=0.98 requirements above need to simultaneously be met with once testing, no
Then this experiment is invalid, need to re-start.
The inspection for the kit that the embodiment of the present invention 3 is provided is determined by reference to the research experiment of value according to above-mentioned detection method
Survey lower limit is 10IU/ml, and internal reference Ct reference values are 40.
Points for attention：
1st, the target molecule of pcr amplification product middle and high concentration can pollute whole lab space in the form of an aerosol, all need
Strictly pcr amplification product is post-processed, due to PCR high sensitivity, it might even be possible to detect 10 in reaction system
The template concentrations of individual copy, so as to bring serious false positive to experiment from now on；
2nd, brief centrifugation is that the aerosol in centrifuge tube is centrifuged into solution in extraction process, need not be examined in operating process
Consider centrifugal force, brief centrifugation；
3rd, centrifuge tube must be covered tightly when mixing by being shaken in extraction process, prevented solution from spilling, caused the dirt of other samples
Dye.
Comparative example 1
This comparative example provides a kind of kit, as different from Example 3, the magnetic bead in extracts reagent in the kit
Not connected target nucleotide sequences.
Comparative example 2
This comparative example provides a kind of detection method, and as different from Example 4, step takes 160ul extracts reagents in (3)
Solution 2 be added in centrifuge tube, concussion mix, brief centrifugation, open centrifugation lid, other be the same as Examples 4.
The specificity experiments of test example 1
Test method：According to the detection method described in the kit embodiment 4 provided using embodiment 3 to eight bases of HBV
Because of type standard items, four HBV positive serums and hepatitis A virus, HCV, Epstein-Barr virus and cytomegalovirus
Know that sample is detected, examine or check the specificity of kit of the present invention, the amplification curve of acquisition as depicted in figs. 1 and 2, by Fig. 1 with
Amplification curve shown in Fig. 2 understands that kit of the present invention is capable of specific detection HBV eight genotype.
The sensitivity experiment of test example 2
Test method：The kit provided using embodiment 3 is according to the detection method described in embodiment 4 to 20 standards
Sample (including 1 negative internal reference product) and 4 standard items are detected that the software carried using instrument is analyzed, and is drawn
The amplification curve of difference detection sample, as a result as shown in Fig. 3, Fig. 4, Fig. 5, this result shows the effective of this sensitivity test
Property.
2.5IU/ml and 10IU/ml standard items are detected respectively using the detection method described in embodiment 4, each
The standard items of concentration detect 100 parts, as a result count as shown in table 4 below：
Table 4
| IU/ml | Detected | Not Detected | Detected (%) |
| 0 | 0 | 0 | 0 |
| 2.5 | 91 | 9 | 91 |
| 10 | 100 | 0 | 100 |
From the result shown in above-mentioned table 4, kit of the present invention detects that sensitivity is high to HBV's, lowest detection line
Reach 10IU/ml positives.
The contrast clinical detection experiment of test example 3
Test method：It is divided into tetra- groups of A, B, C, D to detect 40 parts of clinical samples (containing negative sample), to detect result
Its sensitivity of progress statistical comparison, wherein A groups are the kits provided using the embodiment of the present invention 3 according to described in embodiment 4
Detection method is detected；B groups are the HBV fluorescence quantitative PCR detections examinations developed using Hunan Shengxiang Biological Technology Co., Ltd.
Agent box is detected according to the detection method described in embodiment 4；C groups are according to embodiment 4 using the kit described in comparative example 1
Described detection method is detected；D groups are the kit provided using embodiment 3 according to the detection method described in comparative example 2
Detected, it is as a result as shown in table 5 below：
Table 5
Statistical result is as shown in table 6 below：
Table 6
| Group | Total sample/ | Positive/ | Negative/ | Recall rate % |
| A | 40 | 31 | 9 | 77.5 |
| B | 40 | 26 | 14 | 65 |
| C | 40 | 19 | 21 | 47.5 |
| D | 40 | 21 | 19 | 52.5 |
The limited public affairs of the holy Hunan biotechnology of kit and Hunan provided by the present invention are provided as the result shown in above-mentioned table 6
The recall rate that 40 parts of clinical samples are detected is compared respectively for the agent box of department's exploitation, can effectively verify this kit
With higher sensitivity；The kit that the kit and comparative example 1 provided by the present invention is provided is respectively to 40 parts of clinical samples
The recall rate that this progress is detected is compared, it was demonstrated that the side that kit of the present invention passes through the connection target nucleotide sequences on magnetic bead
Formula, improves the specificity and sensitivity detected to HBV；By the ratio that recall rate is carried out with the detection method that comparative example 2 is provided
Right, the present invention is during hbv nucleic acid is extracted, and the processing mode cooled afterwards using first heating up improves detection
Specificity and sensitivity.
Specification nucleotides and amino acid sequence table
<110>Sixth Man people hospital of Zhengzhou City
<120>One kind extracts hbv nucleic acid with magnetic bead, extracts reagent, extracting method, quantitative detection kit
<160>9
<210>1
<211>33
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, to target linking objective nucleotide sequence.
<400>1
AGACCACCAA ATGCCCCTAT CCTATCA ACA CTT 33
<210>2
<211>29
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, to target linking objective nucleotide sequence.
<400>2
GAGACCTTCG TCTGCGAGGC GAGGAGTT 29
<210>3
<211>24
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, to target linking objective nucleotide sequence.
<400>3
ATCGAGGGTA ATCATTAACG GTGT 24
<210>4
<211>33
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, the primer expanded to PCR.
<400>4
AGACCACCAA ATGCCCCTAT CCTATCA ACA CTT 33
<210>5
<211>29
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, the primer expanded to PCR.
<400>5
GAGACCTTCG TCTGCGAGGC GAGGAGTT 29
<210>6
<211>19
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, the probe expanded to PCR.
<400>6
AGGCAGGTCC CCTAGAAGA 19
<210>7
<211>24
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, the primer expanded to PCR.
<400>7
ATCGAGGGTA ATCATTAACG GTGT 24
<210>8
<211>21
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, the primer expanded to PCR.
<400>8
CGTTCTCAAA CGCCAGCGAA A 21
<210>9
<211>23
<212>DNA
<213>Artificial sequence
<223>Designed according to characteristic nucleotide sequence, the probe expanded to PCR.
<400>9
ACGGTGTGCC AGCATGTGTG GGA 23
Claims (10)
1. one kind extracts hbv nucleic acid magnetic bead, it is characterised in that by magnetic bead one, magnetic bead two and magnetic bead three according to 6：
6：1 ratio mixing composition；The magnetic bead one is the magnetic bead for being connected with target nucleotide sequences one, and magnetic bead two is to be connected with target nucleotide
The magnetic bead of sequence two, magnetic bead three is the magnetic bead for being connected with target nucleotide sequences three；
The target nucleotide sequences one are
5’-Biotin-AGACCACCAAATGCCCCTATCCTATCAACACTT-3’；
Target nucleotide sequences two are
5’-Biotin-GAGACCTTCGTCTGCGAGGCGAGGGAGTT-3’；
Target nucleotide sequences three are
5’-Biotin-C12-ATCGAGGGTAATCATTAACGGTGT-3’。
2. one kind extracts hbv nucleic acid extracts reagent, it is characterised in that by solution 1, solution 2, solution 3 and solution
4 compositions；
Wherein solution 1 by 0.1mol/L EDTA-Na2Solution, 2mol/L NaOH solution, 6mol/L guanidine hydrochloride solution, quality
The tween solution that concentration is 20% is according to 8：8：40：21 volume ratio mixing composition；
Solution 2 by 4mol/L HCl solution and concentration for 2.5ug/ul magnetic bead solution according to 32：1.3 volume ratio mixing group
Into；The magnetic bead is magnetic bead as claimed in claim 1；
Solution 3 by 0.1mol/L EDTA-Na2Solution, 4mol/L NaCl solution, 1mol/L Tris-HCl buffer solutions and water
According to 0.25：0.625：0.25：49 volume ratio mixing composition；
Solution 4 by solution 3 and mass concentration for 20% tween according to 20：1 volume ratio mixing composition.
3. a kind of extracting method for extracting hbv nucleic acid, it is characterised in that including following operating procedure：1) will be to be measured
Sample is added in centrifuge tube, then takes the solution 1 in the extracts reagent described in claim 2 to add in centrifuge tube, vibration is mixed,
Brief centrifugation；
2) take the solution 2 in the extracts reagent described in claim 2 to add in centrifuge tube, after vibration is mixed, add at a temperature of 50 DEG C
Heat is after 10 minutes, brief centrifugation, and room temperature is cooled down 2 minutes；
3) by step 2) centrifuge tube after processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandon supernatant, add claim 2
Solution 4 in described extracts reagent, vibration is mixed, brief centrifugation；
5) by step 3) centrifuge tube after processing is placed on magnetic frame and collects magnetic bead 10 minutes, abandon supernatant, add claim 2
Solution 3 in described extracts reagent, after standing 1 minute, abandons supernatant, that is, completes.
4. a kind of hepatitis type B virus, which quantitatively detects, uses kit, it is characterised in that extract examination including as claimed in claim 1
Agent, PCR reaction solutions, negative quality-control product, positive quality control product and standard items.
5. hepatitis type B virus as claimed in claim 4, which quantitatively detects, uses kit, it is characterised in that the PCR reaction solutions
It is made up of the mixing of following solution：10 × PCR reactions buffer solution, 2mmol/LdNTPs, mass concentration are sub- for 50% dimethyl
Sulfolane solution, 4mol/L alkali solution of beet, 30pmol/ul target nucleotides primer mixed liquor, 30pmol/ul target nucleotide probes are molten
Liquid, 30pmol/ul interior label primers mixed liquor, 30pmol/ul internal standard probes solution, 1U/ μ l hot resistant DNA polymerases, 1U/ μ l urine are phonetic
Pyridine DNA glycosylases, water.
6. hepatitis type B virus as claimed in claim 5, which quantitatively detects, uses kit, it is characterised in that the PCR reaction solutions
Dimethyl sulphoxide solution, 4mol/L beets by 10 × PCR reactions buffer solution, 2mmol/L dNTPs, mass concentration for 50%
Indexed in aqueous slkali, 30pmol/ul target nucleotides primer mixed liquor, 30pmol/ul target nucleotides probe solution, 30pmol/ul
Thing mixed liquor, 30pmol/ul internal standard probes solution, 1U/ μ l hot resistant DNA polymerases, 1U/ μ l uracil dna glycosylases and water
According to 5：5：2：1：2.5：1：1：1：1.2：1：29.3 volume ratios mixing composition.
7. the hepatitis type B virus as described in claim 5 or 6, which quantitatively detects, uses kit, it is characterised in that the 30pmol/
Contain target nucleotide forward primer and target nucleotide reverse primer in L target nucleotide primer mixed liquors；30pmol/L interior label primers
Contain internal standard forward primer and internal standard reverse primer in mixed liquor；
Its target nucleotide forward primer sequence is：
5’-AGACCACCAAATGCCCCTATCCTATCAACACTT-3’；
Target nucleotide reverse primer sequences are：
5’-GAGACCTTCGTCTGCGAGGCGAGGGAGTT-3’；
Internal standard forward primer sequence is：5’-ATCGAGGGTAATCATTAACGGTGT-3’；
Internal standard reverse primer sequences are：5’-CGTTCTCAAACGCCAGCGAAA-3’；
Target nucleotide probe sequence is：5 '-AGGCAGGTCCCCTAGAAGA-3 ', the end of target nucleotide probe 5 ' flag F AM fluorescence
Reporter group, 3 ' end mark MGB fluorescent quenching groups；
Internal standard probe sequence is：5 '-ACGGTGTGCCAGCATGTGTGGGA-3 ', internal standard probe 5` end mark HEX fluorescence reports
Group, 3` ends mark BHQ1 fluorescent quenching groups.
8. the hepatitis type B virus as described in any one of claim 4~6, which quantitatively detects, uses kit, it is characterised in that described
Negative quality-control product is the pUC57-T carriers without hepatitis B virogene.
9. the hepatitis type B virus as described in any one of claim 4~6, which quantitatively detects, uses kit, it is characterised in that described
Positive quality control product is 1.0x103The pUC57-T carriers of copy/ml groups containing hepatitis B virogene target nucleotide sequences.
10. the hepatitis type B virus as described in any one of claim 4~6, which quantitatively detects, uses kit, it is characterised in that described
Standard items are the pUC57-T restructuring containing 109 base pair nucleotide fragments at hepatitis B virus core gene terminal conserved sequences
Plasmid, the plasmid is bred in escherichia coli DH5a, is specifically included：
Working standard 1：Contain 1.0x107Copy/ml target nucleotide sequences recombinant plasmid；
Working standard 2：Contain 1.0x106Copy/ml target nucleotide sequences recombinant plasmid；
Working standard 3：Contain 1.0x105Copy/ml target nucleotide sequences recombinant plasmid；
Working standard 4：Contain 1.0x104Copy/ml target nucleotide sequences recombinant plasmid.
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