CN105200018A - Pseudovirions and preparation method and application thereof - Google Patents
Pseudovirions and preparation method and application thereof Download PDFInfo
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- CN105200018A CN105200018A CN201510712786.4A CN201510712786A CN105200018A CN 105200018 A CN105200018 A CN 105200018A CN 201510712786 A CN201510712786 A CN 201510712786A CN 105200018 A CN105200018 A CN 105200018A
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Abstract
The invention discloses pseudovirions; a capsid protein of MS2 bacteriophage is wrapped with a DNA-protein complex of a detection sequence formed by series connection of three base fragments of influenza A virus, influenza B virus and beta-actin successively. The invention also provides a preparation method of the pseudovirions and an application of the pseudovirions as quality control products for detecting the influenza A virus and the hepatitis B virus. The preparation method of the pseudovirions is simple and is easy to operate; the obtained pseudovirus is high in purity, can be used as the quality control products in an influenza A virus and influenza B virus nucleic acid detection kit, and has the characteristics of no infectivity, good stability, ribonuclease resistance and the like.
Description
Technical field
The invention belongs to pathogenic agent vitro diagnostic techniques field, be specifically related to a kind of pseudovirion and its preparation method and application.
Background technology
Along with developing rapidly of Protocols in Molecular Biology, various diagnostic technique in molecular biology based on polymerase chain reaction, because it has high specificity, highly sensitive, reproducible, quantitatively accurately, the advantage such as convenient and swift, become the research means of most worthy in biomedical sector, be widely used in the detection of clinical samples.In RNA viruses RT-PCR detects, random error in experimenter's operation, residual, the concentration of target nucleic acid to be amplified, the efficiency of reverse transcription and the reagent problem etc. of inhibition after the difference of temperature, sample nucleic acid extraction between amplification instrument hole, all may affect the efficiency of pcr amplification, and then cause the deviation of result.Nucleic acid amplification detects expects reliable result, quality control product must be used to carry out strict quality control, namely need specific quality control product.Quality Control thing must possess following characteristics: (1) easily prepares; (2) good stability in storage and use procedure; (3) lifeless matter infectivity; (4) whole testing process can be monitored, reliable results.
Cause virus mainly influenza A virus and the Influenza B virus of human world influenza pandemic.A type and Influenza B virus Jun Shu orthomyxoviridae family, its genome is all made up of the minus strand single-stranded RNA of 8 sections.Its structure forms primarily of three parts: 1. core: be positioned at viral innermost layer, is made up of nucleic acid and nucleoprotein.2. stromatin (M albumen): under being positioned at viral lipid coating, and with membrane-bound albumen.3. coating: be positioned at the lipid bilayer outside stromatin, derive from host cell membrane, it includes two kinds of important glycoprotein, has important antigenicity: i.e. hemagglutinin (HA) and neuraminidase (NA).According to the difference of HA and NA protein antigenicity, influenza A virus can be divided into 16 H hypotypes (H1-H16) and 9 N hypotypes (N1-N9) at present.Influenza virus can infect all various types of cells of respiratory tract, and can within it copy, and its main mechanism caused a disease is the cell injury that causes of virus replication and death.
The infection risk potential due to natural viral and the unstable of RNA molecule thereof, the quality control product of current application is as virion, cDNA, plasmid DNA, the exposed RNA and in-vitro transcription RNA of deactivation, all be difficult to reach test requirements document, so develop stable, the communicable quality control product of lifeless matter, not only the RT-PCR of RNA viruses is detected, and all significant to the evaluation of commercial virus RT-PCR test kit.
Virus-like particle is the hollow bead of the one or more structural protein containing certain virus, not containing viral nucleic acid, can not self-replicating, but granular structure can be assembled into, its structure and epitope and natural virion quite similar, there is very strong immunogenicity and well security.Virus-like particle and recombinant plasmid dna are merged by special methods in vitro, the recombinant RNA be transcribed into wraps in virus-like particle, namely forms pseudovirus.Pseudovirus has simulates real virus structure, can resist nuclease degradation, stable, security advantages of higher, provides a kind of strong instrument to the work of current RNA viruses detect delay.MS2 phage is single strand positive strand RNA viruses, is 20 bodies that 26nm is long, has 3569 Nucleotide, include 3 genes, 4 kinds of protein molecules such as encoding mature zymoprotein, envelope protein, replicase protein and crack protein.Phage is made up of 180 envelope protein monomers, a part maturing enzyme albumen and a part geneome RNA, in the research of MS2 phage envelope protein, find that envelope protein and phage replication enzyme 5 ' are held by 19 bases (19, mer) the loop-stem structure RNA sequence formed has specificity to interact, this effect can cause the assembling of phage ghost, meanwhile, phage genome RNA is packaged in coating.
Summary of the invention
The technical issues that need to address of the present invention there is provided a kind of pseudovirion detecting fragment containing series connection influenza A virus, Influenza B virus and β-actin three kinds; This pseudovirus has simulates real virus structure, can resist nuclease degradation, stable, security advantages of higher, and is easy to preparation, storage and transport, can be used as quality control product in influenza A virus and Influenza B virus kit for detecting nucleic acid.The method has the technique effects such as quick, objective and accurate.
The technical scheme solved the problems of the technologies described above is as follows.
A kind of pseudovirus, it is the DNA-protein complexes having been wrapped up the detection sequence be in series by influenza A virus, Influenza B virus and β-actin three kinds of base fragments successively by the capsid protein of MS2 phage.
Wherein in an embodiment, the base fragment of described influenza A virus be SEQIDNO:3 or the replacement of one or more base is carried out to SEQIDNO:3, nucleotide sequence that disappearance, interpolation are modified.
Wherein in an embodiment, the base fragment of described Influenza B virus be SEQIDNO:4 or the replacement of one or more base is carried out to SEQIDNO:4, nucleotide sequence that disappearance, interpolation are modified.
Wherein in an embodiment, the base fragment of described β-actin be SEQIDNO:5 or the replacement of one or more base is carried out to SEQIDNO:5, nucleotide sequence that disappearance, interpolation are modified.
Another object of the present invention is to provide a kind of preparation method of above-mentioned pseudovirus.
The technical scheme realizing this object is as follows.
A preparation method for above-mentioned pseudovirus, comprises the following steps:
(1) design is for the primer of MS2 phage genome, carries out the product that RT-PCR amplification obtains about 1780bp, comprises the envelope protein gene of MS2 phage, maturing enzyme gene, packaging signal sequence;
(2) amplified production is inserted in prokaryotic expression carrier pSE380, construction recombination plasmid pSE380-MS2;
(3) obtained recombinant plasmid pSE380-MS2 is transformed in competent escherichia coli cell, enlarged culturing, extracts plasmid;
(4) obtain containing successively by influenza A virus, second; The nucleotide sequence of the detection sequence that type influenza virus and β-actin three kinds of base fragments are in series, the two ends of this nucleotide sequence are with the restriction enzyme site of restriction enzyme BglII and HindIII;
(5) nucleotide sequence that step (4) obtains is connected with recombinant plasmid pSE380-MS2, construction recombination plasmid pSE380-MS2-AB;
(6) expression and purification of recombinant plasmid pSE380-MS2-AB is induced; Obtain.
Wherein in an embodiment, be SEQIDNO:1 and SEQIDNO:2 for the primer of MS2 phage genome in step (1).
Wherein in an embodiment, step (4) is: with amplimer SEQIDNO:7 and SEQIDNO:8, be that template increases with SEQIDNO:6, the amplified production obtained is described nucleotide sequence.
Wherein in an embodiment, the abduction delivering in step (6) and purifying comprise the following steps:
6.1, by the positive bacterium colony of pSE380-MS2-AB recombinant plasmid, the LB solid medium coated containing ammonia benzyl mycin is dull and stereotyped;
6.2, picking list colony inoculation is in the LB liquid nutrient medium containing ammonia benzyl mycin;
6.3, when to be cultured to A600nm be 0.5-1.0, add IPTG (isopropyl-β-D-thiogalactoside(IPTG)), abduction delivering forms virion;
6.4, adopt sonioation method cracking to go out virus-like particle thing, and add rnase and deoxyribonuclease I by exposed RNA and DNA degradation;
6.5, collected by centrifugation supernatant, qualification, preserves virus-like particle correct for qualification.
Another object of the present invention is to provide the application of above-mentioned pseudovirus.
The technical scheme realizing above-mentioned purpose is as follows.
The application of the external standard quality control product during above-mentioned pseudovirus detects as influenza A virus and/or Influenza B virus.
Wherein in an embodiment, described quality control product is robust positive control during influenza A and/or Influenza B virus detect and critical positive control.
Another object of the present invention there is provided a kind of influenza A virus and/or Influenza B virus kit for detecting nucleic acid.
The technical scheme realizing above-mentioned purpose is as follows.
Influenza A virus and/or Influenza B virus kit for detecting nucleic acid, include detection reagent and external standard quality control product, and described external standard quality control product is above-mentioned pseudovirion.
In another embodiment of the present invention, the detection reagent of described test kit comprises nucleic acid extracting reagent, and such as described nucleic acid extracting reagent comprises nucleic acid extraction liquid A, B, C and Sample dilution; Nucleic acid amplification agents, described nucleic acid amplification agents comprises Inf.A/Inf.B/ β-actin amplification reaction solution, Taq DNA polymerase and M-MLV ThermoScript II;
In addition, working instructions can also be included.
Described external standard quality control product is robust positive control, faces positive control; Robust positive control, facing positive control is above-mentioned pseudovirion.
The present invention devises a kind of pseudovirion of the quality control product for influenza A virus, Influenza B virus newly, this pseudovirion contains the DNA-protein complexes having wrapped up the detection sequence be in series by influenza A virus, Influenza B virus and β-actin three kinds of base fragments successively, the pseudovirion of fragment should be detected containing series connection influenza A virus, Influenza B virus and β-actin three kinds, there is the advantage of the following aspects: 1, without infectivity through this virus-like particle of verification experimental verification, when preparing and use to personnel and environment all without danger; 2, good stability, the enzyme of resistance to RNA; 3, carrying out omnidistancely to detect whole experimentation in nucleic acid extraction and PCR qualification process.The present invention can be used as the quality control product in influenza A virus and Influenza B virus kit for detecting nucleic acid, has very strong practical value.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates of plasmid pSE380 in embodiment 1;
Fig. 2 is the fluorescent PCR detected result of robust positive control and critical positive control homogeneity test in embodiment 2;
Fig. 3 is the fluorescent PCR detected result of robust positive control and critical positive control stability test in embodiment 3;
Fig. 4 is the first time fluorescent PCR detected result of 5 parts of clinical samples in embodiment 4;
Fig. 5 is the second time fluorescent PCR detected result of 5 parts of clinical samples in embodiment 4.
Embodiment
For the ease of understanding the present invention, will be described more fully the present invention below.The present invention can realize in many different forms, is not limited to embodiment described herein.On the contrary, provide the object of these embodiments be make the understanding of disclosure of the present invention more comprehensively thorough.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.
Unless otherwise defined, all technology used in the present invention and scientific terminology are identical with belonging to the implication that those skilled in the art of the present invention understand usually.The object of the term used in specification sheets of the present invention just in order to describe specific embodiment, is not used in restriction the present invention.Term "and/or" used in the present invention comprises arbitrary and all combinations of one or more relevant Listed Items.
Embodiment 1
The present embodiment detects the preparation method of the pseudovirion of fragment containing series connection influenza A virus, Influenza B virus and β-actin three kinds, and its concrete preparation process is:
(1), the structure of recombinant plasmid pSE380-MS2:
1, according to the MS2 phage gene sequence (NC_001417) in GenBank database, autonomous design is used for pcr amplification and comprises the envelope protein gene of MS2 phage, maturing enzyme gene, the primer of packaging signal sequence synthetic; Primer is: MS2L:CCTTTCGGGGTCCTGCTC (SEQIDNO:1) MS2BglIIR:GATTAGATCTGAGTTGAACTTCTTTGTTGTCTTC (SEQIDNO:2).
2, apply above-mentioned primer, with MS2 phage gene for masterplate, technology carries out the product obtaining about 1780bp of RT-PCR amplification routinely, comprises the envelope protein gene of MS2 phage, maturing enzyme gene, packaging signal sequence.
3, the MS2 gene fragment obtained increasing and plasmid pSE380 (Fig. 1 asked for an interview by collection of illustrative plates) use restriction enzyme NcoI and BglII double digestion respectively, then both are connected by T4DNA ligase enzyme, obtain connecting product;
4, the connection product conversion that above-mentioned steps 3 obtains is entered in competent escherichia coli cell, choose single bacterium colony and carry out bacterium colony PCR qualification, the correct single bacterium colony of qualification is carried out enlarged culturing and extracts plasmid, by recombinant plasmid called after pSE380-MS2, is placed in-20 DEG C of preservations.
(2), the structure of recombinant plasmid pSE380-MS2-AB:
1, according to the detection fragment resultant string connection sequence of influenza A virus, Influenza B virus and β-actin:
GAGGTCGAAACGTATGTTCTCTCTATCGTTCCATCAGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTGAAGA(SEQIDNO:3)
CCCTGCTTGCTCGTAGTATGGTCGTTGTTAGGCCCTCTGTGGCGAGCAAAGTGGTGCTTCCCATAAGC(SEQIDNO:4)
TGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGC(SEQIDNO:5)。
The detection sequence formed after its series connection is
GAGGTCGAAACGTATGTTCTCTCTATCGTTCCATCAGGCCCCCTCAAAGCCGAGATCGCGCAGAGACTTGAAGACCCTGCTTGCTCGTAGTATGGTCGTTGTTAGGCCCTCTGTGGCGAGCAAAGTGGTGCTTCCCATAAGCTGGCACCCAGCACAATGAAGATCAAGATCATTGCTCCTCCTGAGCGCAAGTACTCCGTGTGGATCGGC(SEQIDNO:6)。
2, autonomous design the pcr amplification primer pair of synthetic tandem sequence, sequence is:
ABRF:cgggatccGAGGTCGAAACGTATGTTCTCT(SEQIDNO:7)
ABRR:ggtaaagcttctgcagggtaccagatctGCCGATCCACACGGAGTACTTG(SEQIDNO:8)。
With above-mentioned detection sequence (SEQIDNO:6) for amplification template, technology carries out pcr amplification routinely.
3, the series connection fragment obtained increasing and recombinant plasmid pSE380-MS2 use restriction enzyme BglII and HindIII double digestion respectively, then both are connected by T4DNA ligase enzyme, obtain connecting product.
4, the connection product conversion that above-mentioned steps 3 obtains is entered in competent escherichia coli cell, choose single bacterium colony and carry out bacterium colony PCR qualification respectively, the correct single bacterium colony of qualification is carried out enlarged culturing and extracts plasmid, by recombinant plasmid called after pSE380-MS2-AB.
(3), abduction delivering and purifying:
Above-mentioned steps two is identified the correct positive bacterium colony containing pSE380-MS2-AB recombinant plasmid, recoat the LB solid medium be distributed in containing ammonia benzyl mycin dull and stereotyped, picking list colony inoculation is in the LB liquid nutrient medium containing ammonia benzyl mycin, when to be cultured to A600nm be 0.5-1.0, adding IPTG to final concentration is 1.0mmol/L, continues to cultivate 36h.Then adopt sonioation method cracking to go out virus-like particle thing, in solution, add rnase and deoxyribonuclease I is 1mg/L to final concentration, 37 DEG C of water-bath effect 2h.Collected by centrifugation supernatant, is the present invention detects fragment pseudovirion containing series connection influenza A virus, Influenza B virus and β-actin three kinds, 4 DEG C of preservations.
Describedly obtain pseudovirion, it is the DNA-protein complexes having been wrapped up the detection sequence (SEQIDNO:6) be in series by influenza A virus, Influenza B virus and β-actin three kinds of base fragments (being followed successively by SEQIDNO:3, SEQIDNO:4, SEQIDNO:5) successively by the capsid protein of MS2 phage.
(4), the preparation of robust positive control and critical positive control:
1, carry out pre-dilution experiment with the virus-like particle of viral dilution liquid gained, measure its CT value;
2, according to pre-dilution experiment, virus-like particle stoste is carried out diluting and detecting;
3, Ct value drops between 18.0-21.0 and is robust positive control product, is diluted to Ct value and drops between 27.0-30.0 and be critical positive control;
4,220 μ L often pipe packing be stored in-20 DEG C.
Embodiment 2: virus-like particle is as the uniformity test of robust positive control and critical positive control
Get-20 DEG C of robust positive control and each 10 of critical positive control preserved, the centrifugal rear 50uL that respectively gets of concussion mixing carries out fluorescent PCR detection, amplification system and amplification program and primer and probe as follows.
RT-PCR amplification system:
| 5 × RT-PCRbuffer (final concentration) | 1× |
| DNTP (final concentration) | 0.6mM |
| Primer (usage quantity) | 10pmol |
| Probe (usage quantity) | 3pmol |
| Taq (usage quantity) | 1U |
| MMLV (usage quantity) | 50U |
| DEPC water | Complement to 20 μ l |
| (template) | (5μl) |
RT-PCR response procedures is as follows:
Primer and probe as follows:
The Ct value of fluorescent PCR result takes statistics the homogeneity of analysis, assessment robust positive control and critical positive control, and experimental result is shown in accompanying drawing 2, and data preparation is as following table:
We adopt relative standard deviation and the variation coefficient to evaluate homogeneity, represent with CV:
Mean value X=(Ct1+Ct2+Ct3++Ct9)/9
Standard deviation: α
In formula, n is check sum
CV=standard deviation/mean value
Qiang Yang: CV (IA)=0.1816/18.41=0.99%; CV (IB)=0.26387/18.4=1.43%; CV (β-actin)=0.1675/18.34=0.91%
Face sun: CV (IA)=0.21458/27.84=0.77%; CV (IB)=0.24436/27.89=0.88%; CV (β-actin)=0.18531/27.94=0.66%
Result shows, each CV value that Qiang Yangyu faces sun is all less than 5%, and between each pipe, difference is not remarkable, has good uniformity.
Embodiment 3: virus-like particle is as the stability analysis of robust positive control and critical positive control
Different time points gets-20 DEG C of robust positive control preserved and each 3 pipes of critical positive control carry out fluorescent PCR mensuration, and detection method and the primer and probe are see embodiment 2.And carry out the stability of each preservation condition of statistical study.Once, beginning in the 6th month monthly samples once, and carries out fluorescent PCR mensuration, statistical study in sampling in 1st month.Accompanying drawing 3 is the result figure of stability test in October.10 times stability test data results sees the following form:
Robust positive control product detect the analysis of variance in regression of CT value (IA):
Robust positive control product detect the analysis of variance in regression of CT value (IB):
Robust positive control product detect the analysis of variance in regression of CT value (β-actin):
Face the analysis of variance in regression that positive reference substance detects CT value (IA):
Face the analysis of variance in regression that positive reference substance detects CT value (IB):
Face the analysis of variance in regression that positive reference substance detects CT value (β-actin):
Significance P>0.05, show each sample time robust positive control and critical positive control change difference not obvious, in 14 months ,-20 DEG C have good stability, therefore its quality guaranteed period is decided to be 12 months is feasible.
Embodiment 4: the application of robust positive control and critical positive control
One, specimen material and plant and instrument:
Influenza A virus and Influenza B virus kit for detecting nucleic acid (PCR-fluorescence probe method, probe and primer are as described in Example 2), 5 parts of clinical samples, 0.6ml centrifuge tube, suction pipette head, whizzer (joining 0.6ml rotor or adapter).Recommendation import centrifuge tube and band filter core suction pipette head operate, and do not use the disposable emulsion gloves of band fluorescent substance in testing process.
Two, nucleic acid extraction:
Nucleic acid extracting reagent:
Institute all can at room temperature carry out in steps.As whizzer has refrigerating function, please temperature is set in 25 DEG C.Detailed operation process is as follows:
1, get 200 μ l nucleic acid extraction liquid A in n (n=sample number+3 external standard quality control products) individual 0.6ml centrifuge tube, often pipe adds mark (negative control need not add interior mark) in 5 μ l.
2, often pipe adds sample to be checked, negative control, robust positive control, each 50 μ l of critical positive control respectively, performs mark, and abundant mixing is also of short duration centrifugal, and room temperature leaves standstill 3 minutes;
3, often pipe adds 200 μ l nucleic acid extraction liquid B, fully mixes, centrifugal 10 minutes of 13000rpm, inhales and abandons supernatant;
4, often pipe adds 200 μ l nucleic acid extraction liquid C, puts upside down for several times mixing, centrifugal 5 minutes of 13000rpm, inhales and abandon supernatant, uncaps within 2 ~ 5 minutes, to dry in room temperature or 55 DEG C of placements;
5, often pipe adds 25 μ l Sample dilution, lid upper tube cap, and with pointing bottom bomb tube or vibrator concussion, nucleic acid is fully dissolved, and the of short duration centrifugal liquid that makes all falls within bottom pipe, and on ice or 4 DEG C of placements, detect for RT-PCR.
Three, RT-PCR amplification:
RT-PCR amplification influenza A virus, Influenza B virus and strong sun face positive reference substance amplified reaction, and detection method is see embodiment 2.
1, dosing (carrying out in reagent area in preparation): take out amplification reaction solution, room temperature is fully mixing after melting, of short duration centrifugal, and to extract reaction solution with enzyme according to following proportioning configuration reaction solution respectively by reaction number n (n=sample number+3 external standard quality control products):
Calculate each reagent dosage, add proper volume in a centrifuge tube, fully mix, of short duration centrifugal, be then dispensed in transparent PCR eight comb or 96 hole PCR Sptting plates by 20 μ l/ pipes, be transferred to sample process district;
2, application of sample (carrying out in sample process district): add the sample handled well in 5 μ l step 2 respectively, builds pipe lid (transparent flat cover), is transferred to nucleic acid amplification district;
3, RT-PCR amplification (carrying out in nucleic acid amplification district):
Reaction tubes/plate is placed in the full-automatic quantitative real time PCR Instrument sample cell of ABIPrism7500 by 3.1.
3.2 open instrument configuration software (for v1.4 version software), new files.
3.3Setup window is arranged: choose placed sample well, click right opens WellInspector window, and (ReporterDye:TEXASRED (TEX)/FAM/JOE tri-kinds chooses simultaneously to add fluorescence probe marking signal by AddDetector button; QuencherDye:None.Wherein TEX fluorescence instruction influenza A virus, FAM fluorescence instruction Influenza B virus, mark β-actin in the instruction of JOE fluorescence.); According to correspondence position input amendment information in SampleName hurdle, negative control (NTC), unknown sample (Unknow), positive control (Unknow) are set in Task hurdle.
3.4Instrument window is arranged:
Cycling condition: 48 DEG C 5 minutes,
94 DEG C 2 minutes,
94 DEG C 10 seconds, 55 DEG C 35 seconds, 40 circulations.
SampleVolume is set to 25 μ l.
DataCollection：Stage3，Step2(55.00：35)
3.5 preserve file, working procedure.
Four, interpretation of result:
1, reaction preserves data file after terminating immediately.
2, analysis condition is arranged: according to the start value of image adjustment Baseline after analyzing, end value (can according to test situation from Row sum-equal matrix, generally start value variable range in 1 ~ 10, end value variable range 5 ~ 20).The Value value of Threshold is set at Linear collection of illustrative plates window, or direct pull threshold line, make the setting of value higher than background fluorescence signal, and be positioned at the amplification curve exponential amplification phase.Select Analyze automatic analysis result.
3, to Results/Report window record sample Ct value.
Five, quality control:
1, negative control all without rising appreciably, need there is no obvious S type amplification curve by fluorescent signal under TEX, FAM and JOE tri-kinds of fluoresceins;
2, robust positive control all need rise appreciably by fluorescent signal under TEX, FAM and JOE tri-kinds of fluoresceins, have typical S type curve, and Ct value should between 18.00 ~ 21.00;
3, critical positive control all need rise appreciably by fluorescent signal under TEX, FAM and JOE tri-kinds of fluoresceins, have typical S type curve, and Ct value should between 27.00 ~ 30.00.
4, under requiring the same fluorescein in same experiment, identical Threshold is set, and meets above three Quality Control requirements.Otherwise experimental result is false.
5, the interior mark reaction of all clinical oropharynx swab sample should be all positive, and namely Ct value is less than or equal to 35.00, otherwise this sample results is false.
[reference value (term of reference)]
By the test to a large amount of clinical feminine gender and positive sample, the reference Ct value of this test kit is 35.00 to utilize ROC curve method to determine.Namely 0<Ct value≤35.00 of sample to be checked are positive, otherwise are negative.35.00≤Ct value≤38.00 are test kit gray area, and the sample being in gray area rechecks result for positive or be still in gray area and be all judged to the positive, meet negative findings discrimination standard then sample for negative.
[explanation of assay]
Every data that composite analyser provides, set rational threshold value (Threshold) and baseline (Baseline), make instrument provide correct result.Please judge separately for TEX, FAM or JOE fluorescence respectively during interpretation of result.
One, Quality Control Analysis:
Each test all needs to detect negative quality control product, robust positive control and critical positive control, can carry out the judgement of detected result when quality control product result meets quality control requirement in the method for inspection.
Two, result is explained:
1, negative findings judges: sample to be checked is without obvious fluorescence amplification and without typical S type curve, or measuring samples TEX fluorescence (influenza A virus) Ct>38.00 or Undet, FAM fluorescence (Influenza B virus) Ct>38.00 or Undet, JOE fluorescence (interior mark β-actin) Ct≤35.00, be judged to be negative sample.
2, positive findings judges: fluorescence amplification is obvious, have typical S type curve, and Ct value meets lower surface condition, is judged to be positive sample.
3, gray area result judges:
Fluorescent signal has obvious amplification, but TEX fluorescence (Inf.A) 35.00≤Ct value≤38.00, FAM fluorescence (Inf.B) 35.00≤Ct value≤38.00, be test kit gray area, the sample being in gray area needs to recheck herein.According to practical situation, can repeated sampling or by same this duplicate detection of increment.Recheck result for positive or be still in gray area and be all judged to the positive, then sample is for negative to meet negative findings discrimination standard, and in this way, reference symbol closes negative findings discrimination standard, then judge that sampling is defective.During by same this duplicate detection of increment, sample is carried out suitable concentrated and purified, the sensitivity of detection can be improved in theory, but the increase effect of sensitivity is not verified.
4, result example:
Sample to be checked is 5 parts of clinical samples, uses this test kit to detect.Detected result comprises robust positive control, critical positive control, negative control and clinical sample 5, and detected result is shown in accompanying drawing 4 and accompanying drawing 5, and data preparation is following table:
Each technical characteristic of the above embodiment can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this specification sheets is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a pseudovirion, is characterized in that, it is the DNA-protein complexes having been wrapped up the detection sequence be in series by influenza A virus, Influenza B virus and β-actin three kinds of base fragments successively by the capsid protein of MS2 phage.
2. pseudovirion according to claim 1, is characterized in that, the base fragment of described influenza A virus be SEQIDNO:3 or the replacement of one or more base is carried out to SEQIDNO:3, nucleotide sequence that disappearance, interpolation are modified.
3. pseudovirion according to claim 1, is characterized in that, the base fragment of described Influenza B virus be SEQIDNO:4 or the replacement of one or more base is carried out to SEQIDNO:4, nucleotide sequence that disappearance, interpolation are modified.
4. the pseudovirion according to any one of claim 1-3, is characterized in that, the base fragment of described β-actin be SEQIDNO:5 or the replacement of one or more base is carried out to SEQIDNO:5, nucleotide sequence that disappearance, interpolation are modified.
5. a preparation method for pseudovirion, is characterized in that, comprises the following steps:
(1) use the primer for MS2 phage genome, with MS2 phage gene for template, carry out RT-PCR amplification and obtain product, this amplified production comprises envelope protein gene, maturing enzyme gene, the packaging signal sequence of MS2 phage;
(2) amplified production is inserted in prokaryotic expression carrier pSE380, construction recombination plasmid pSE380-MS2;
(3) obtained recombinant plasmid pSE380-MS2 is transformed in competent escherichia coli cell, enlarged culturing, extracts plasmid;
(4) obtain the nucleotide sequence of detection sequence containing being in series by influenza A virus, Influenza B virus and β-actin three kinds of base fragments successively, the two ends of this nucleotide sequence are with the restriction enzyme site of restriction enzyme BglII and HindIII;
(5) nucleotide sequence that step (4) obtains is connected with recombinant plasmid pSE380-MS2, construction recombination plasmid pSE380-MS2-AB;
(6) induce the expression and purification of recombinant plasmid pSE380-MS2-AB, to obtain final product.
6. the preparation method of pseudovirion according to claim 5, is characterized in that, is SEQIDNO:1 and SEQIDNO:2 for the primer of MS2 phage genome in step (1).
7. the preparation method of pseudovirion according to claim 5, it is characterized in that, step (4) is: with amplimer SEQIDNO:7 and SEQIDNO:8, be that template increases with SEQIDNO:6, the amplified production obtained is described nucleotide sequence.
8. the preparation method of the pseudovirion according to any one of claim 5-7, is characterized in that, the abduction delivering in step (6) and purifying comprise the following steps:
(6.1), by the positive bacterium colony of pSE380-MS2-AB recombinant plasmid, the LB solid medium coated containing ammonia benzyl mycin is dull and stereotyped;
(6.2), picking list colony inoculation is in the LB liquid nutrient medium containing ammonia benzyl mycin;
(6.3), when to be cultured to A600nm be 0.5-1.0, add IPTG, abduction delivering forms virion;
(6.4), adopt sonioation method cracking to go out virus-like particle thing, and add rnase and deoxyribonuclease I by exposed RNA and DNA degradation;
(6.5), collected by centrifugation supernatant, qualification, preserves the correct virus-like particle of qualification.
9. the application of the external standard quality control product during the pseudovirion described in any one of claim 1-4 detects as influenza A virus and/or Influenza B virus.
10. influenza A virus and/or an Influenza B virus kit for detecting nucleic acid, is characterized in that, includes detection reagent and external standard quality control product, and described external standard quality control product is the pseudovirion described in any one of claim 1-4.
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