CN101376912A - Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method - Google Patents
Loop-mediated isothermal amplification detection kit of influenza A3 viruses and detecting method Download PDFInfo
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Abstract
The invention provides a loop-mediated isothermal amplification test method of an influenza A3 virus. The method comprises the following steps: a specificity primer sequence is designed according to an influenza A3 virus hemagglutinin gene, a sample RNA to be tested is extracted as the template to have LAMP reaction; the LAMP reaction result is analyzed; if the LAMP amplification result is positive, the sample to be tested contains the influenza A3 virus. The LAMP influenza A3 virus test method is characterized by convenience, economy, fastness, sensitivity and specificity with flexible and convenient result judgment, is applicable in clinical or laboratory diagnosis, and has broad application prospect.
Description
(1) technical field
The present invention relates to a kind of ring mediated isothermal amplification (loop-mediatedisothermal amplification, LAMP) detection kit and detection method of 3 type grippe virus.
(2) background technology
The clinical symptom of influenza (influenza) is easily obscured mutually with common cold, and finally the method for chamber is made a definite diagnosis by experiment.In the laboratory diagnosis means, the most direct the most classical be the gargarism of gathering the patient, inoculated into chick embryo or dog kidney passage cell (MDCK) carry out virus to be separated to finalize the design and determines.But adopt this method usually to need to spend the long time, be difficult to adapt to the needs that break out the epidemic situation quick diagnosis.
(Polymerase Chain Reaction, PCR) birth of technology has promoted the development of nucleic acid detection technique even whole Protocols in Molecular Biology to polymerase chain reaction in 1985 greatly, and day by day becomes the important means in the nucleic acid detection technique.Constantly occurred again subsequently such as reverse transcriptase-polymerase chain reaction (Reverse Transcriptase-PCR, RT-PCR), real-time fluorescence quantitative polymerase chain reaction a series of nucleic acid amplification detection techniques such as (Real Time Fluorescent Quantified PCR), can realize amplification and qualitative and quantitative detection to nucleic acid samples.In recent years, adopt the RT-PCR method that influenza nucleic acids is detected both at home and abroad, classical viral separation method is more responsive fast.Fluorescent quantitative PCR technique has utilized the efficient amplification of round pcr to DNA, and the high specific of probe technique and the susceptibility of spectroscopic techniques and quantitative advantage have obtained good application in cut-and-try work.But, this technology on promoting the use of, basic unit is very restricted because the quantitative fluorescent PCR instrument costs an arm and a leg.
(loop-mediated isothermal amplification, LAMP) technology is the sensitivity of a kind of alternative PCR of going out of development in recent years, special, nucleic acid amplification technologies conveniently to ring mediated isothermal amplification.Its principle be adopt can the specific recognition target sequence on 6 sites 4 primers and a kind ofly have an active archaeal dna polymerase of strand displacement, about 65 ℃, less than the amplification of carrying out nucleic acid in the time of 1h, its amplification efficiency can reach 10
9Individual～10
10Individual copy number magnitude.Produce stem-ring structure in amplified reaction, guarantee that primer and its hybridization start new round nucleic acid and synthesizes, the byproduct of reaction magnesium pyrophosphate precipitates available naked-eye observation and arrives, and increases judging whether.This TRAP has simply, fast, accurately, cheap, easy characteristics such as detection.
The LAMP technology has identical sensitivity with round pcr, but its technology platform is more superior than PCR.Because its reaction is that multiple primer starts jointly, has improved the specificity of reaction result; Because what carry out is isothermal duplication, make to be reflected in the constant water bath box and just can finish, not only saved instrument cost, and made the detection of nucleic acids working method easier, be applicable to that a large amount of samples detect simultaneously.
(3) summary of the invention
The ring mediated isothermal detection kit and the detection method that the purpose of this invention is to provide a kind of quick, easy, economic 3 type grippe virus.
The technical solution used in the present invention is:
A kind of 3 type grippe virus ring mediated isothermal amplification detects detection kit, and described test kit comprises following specific primer sequence:
F3：GCAGAATAAGCATCTATTGGA；
B3：GCCCCATATGTGATCCTG；
FIP：CGTATTTTGAAGTAACCCCKAGGAGGGAGACATACTT
TTGATTA?ACAGC；
BIP：
AAGCTCAATAATGAGRTCAGATGCAGTCATTGGGAATG
CTTCCAT；
LB：GCAATTCTGAATGCATCACTCCA。
Also comprise dNTP, MgSO in addition in this test kit
4, the AMV reversed transcriptive enzyme, Bst-DNApolymerase, Bst-DNA polymerase Buffer etc., these components belong to common practise to those skilled in the art, can select for use as required.
The invention still further relates to the ring mediated isothermal detection method of 3 type grippe virus, described method comprises:
(1) as follows according to 3 type grippe virus hemagglutinin gene design specific primer sequence:
F3：GCAGAATAAGCATCTATTGGA；
B3：GCCCCATATGTGATCCTG；
FIP：CGTATTTTGAAGTAACCCCKAGGAGGGAGACATACTTT
TGATTA?ACAGC；
BIP：
AAGCTCAATAATGAGRTCAGATGCAGTCATTGGGAATG
CTTCCAT；
LB：GCAATTCTGAATGCATCACTCCA；
(2) extracting testing sample RNA, is that sequence F3, B3, FIP, BIP and LB are that primer carries out the LAMP reaction in template, the step (1) with testing sample RNA;
(3) after the LAMP reaction finishes, analyze, if the LAMP reaction result is positive, then testing sample contains 3 type grippe virus.
Key of the present invention is the primer design selection, the LAMP reaction system is formed, reaction conditions is selected and the reaction result judgement all can be undertaken by this area ordinary method, LAMP amplification reaction system of the present invention requires F3, B3, FIP and 4 primers of BIP to mate fully just and can increase, so other external source contaminated nucleic acid in the reaction system or primer are very little to the interference of reaction.By the ring primer of the terminal strand stem ring regional complementarity of design and 2 primer dumbbell shaped structures 5 ', can improve the quantity that DNA synthetic in the LAMP method begins the site, make the LAMP reaction in 30 minutes, to finish, thereby improved the speed of reaction more.
Preferably, LAMP reaction system final concentration of the present invention is composed as follows: F3,0.2 μ mol/L; B3,0.2 μ mol/L; BIP, 1.6 μ mol/L; FIP, 1.6 μ mol/L; LB, 0.8 μ mol/L; DNTP, 1.4mM; MgSO
4, 6mM; The AMV reversed transcriptive enzyme, 0.4U/ μ L; Bst-DNApolymerase, 0.32U/ μ L; Bst-DNA polymerase Buffer, final concentration be 1 *; Sample RNA; Solvent is the DEPC treating water.Described Bst-DNA polymerase Buffer final concentration is 1 *, be meant that the final concentration of each component of damping fluid in reaction system is identical with each component concentrations among 1 * Bst-DNApolymerase Buffer.Usually adopt 10 * Bst-DNA polymerase Buffer of reaction system 1/10 volume.Sample RNA consumption is a conventional amount used in the LAMP reaction system, and those skilled in the art can determine according to actual needs that the RNA sample should comprise 10 copies at least in the LAMP reaction system usually.
Preferably, described LAMP reaction conditions is 60～65 ℃ of reactions 30～60min, 80 ℃ of 2min termination reactions then.
The described method that the LAMP reaction result is analyzed judgement comprises: 1): naked eyes direct viewing reaction turbidity, 2): DNA electrophoresis, 3): turbidity detects in real time; Can also be after adding fluorescence dye by 4): visual inspection reaction solution colour-change, 5): uv irradiating detects fluorescence, 6 down): detect in above-mentioned 6 kinds of methods such as fluorescence one or more in real time.Wherein:
1): naked eyes direct viewing reaction turbidity: when nucleic acid is synthetic in a large number, pyrophosphate ion of separating out from dNTP and the Mg the reaction solution
2+Combination, produce the magnesium pyrophosphate precipitation, and sedimentary amount increases with the increase of amplified production.Qualitative observation: after reaction finished, experimental result can be by the direct interpretation of visual inspection.
2): the DNA electrophoresis: the reaction solution that takes a morsel carries out agarose gel electrophoresis, under ultra violet lamp, occurs typical scalariform band in the EB stained gel, and this pipe amplified reaction positive then is described.
3): turbidity detects in real time: by turbidimeter and software kit, reflect the variation of turbidity in the mixed solution in real time and carry out graphicprocessing, result of determination.
4): 5) and 6) if all be according to containing amplified production after adding fluorescence dye SYBR Green I, reaction mixture becomes green, otherwise the reaction result that keeps the orange constant phenomenon of SYBR Green I to carry out is judged; It is to detect the fluorescence that SYBRGreen I sends by the fluoroscopic examination instrument that real-time fluorescence detects.
Preferably, described determination methods as a result is as follows: after the LAMP reaction finishes, get the LAMP reaction solution and carry out agarose gel electrophoresis, under ultra violet lamp, trapezoid-shaped strips occurs in the EB stained gel, then the LAMP amplification is positive.
Described sample RNA extracts and can carry out according to a conventional method, extracts as adopting Qiagen RNeasy MiniKit or other test kit.
Among the present invention, the extraction step of described sample RNA can be:
1) adds 500 μ l RLT and 6 μ L beta-mercaptoethanols in the centrifuge tube;
2) in above-mentioned solution, add 200 μ L samples (comprising gargarism, throat swab, respiratory tract aspirate etc.), vibration mixing 1min;
3) add isopyknic 70% ethanol mixing;
4) get 700 μ l mixed solutions and add in the interior centrifugal post of collection tube the centrifugal 15s of 12000rpm;
5) repeating step 4 once;
6) add 700 μ l RW1 damping fluids in centrifugal post, the centrifugal 15s of 12000rpm;
7) add 500 μ l RPE damping fluids in centrifugal post, the centrifugal 15s of 12000rpm;
8) repeating step 7 once;
9) liquid in the collection tube to the greatest extent, empty from once;
10) add the water dissolution RNA and the centrifugal collection of no RNA enzyme in right amount, place-70 ℃ of preservations.
The strain that is used for the specificity analyses of LAMP reaction comprises: influenza virus H1/ Beijing/53/97, B/ Shenzhen/12/97 and B/ Beijing/184/9; Edmonston strain Shanghai 191, rubella virus GOS strain, mumps virus Jerry-Lynn strain, respiratory syncytial virus Long strain; Avian influenza virus H 5 N 1 Zhejiang/16/2006, above-mentioned virus utilizes the H3 LAMP primer of design to react after extracting nucleic acid; Through verification experimental verification, adopt detection method of the present invention, have only H3 strains of influenza viruses amplified reaction to be positive, above-mentioned virus strain all is negative, and illustrates that the inventive method has good specificity.
Easy, economic, quick with it, the sensitive and special characteristics of LAMP technology, the determination methods flexible and convenient is suitable for the application in clinical or laboratory diagnosis as a result, and LAMP 3 type grippe virus detection technique provided by the invention has broad application prospects.
Beneficial effect of the present invention is mainly reflected in:
1) easy reaction system: realized carrying out alternately of isothermal duplication link and detection by quantitative link under constant temperature, in the nucleic acid isothermal amplification, realized the accumulation of turbidity or fluorescent signal, entire reaction is finished in an individual system;
2) isothermal amplification system efficiently: the present invention is based on has the isothermal amplification technique that the active Bst archaeal dna polymerase of strand displacement carries out, and is providing under the condition of corresponding reacted constituent, has realized that the index of target gene to be checked increases.When improving amplification efficiency, reduce the cost of detecting instrument, and reduced unnecessary non-specific amplification product pollution in the reaction;
3) detection speed is fast; Nucleic acid detection technique of the present invention will increase and detects two reaction process and merge mutually in a reaction system and finish, and whole process does not have the heating and cooling circulation, has saved the time; Roll the ring primer by design and primer dumbbell structural region complementary simultaneously, can improve the speed of reaction greatly;
4) detection sensitivity height: nucleic acid detection technique of the present invention, can in 0.5～1 hour time, realize 10 of sample
6～10
9Amplification doubly, I is carried out detection by quantitative to the nucleic acid molecule of 10ng, has improved the sensitivity that detects;
5) test set is simple: with respect to other nucleic acid quantification detection technique, the present invention does not need specific reaction and detecting instrument, as long as common constant water bath box (real-time detection also needs turbidometer or fluorescence detector as need) can carry out, be convenient to use and promote.
(4) description of drawings
Fig. 1 is H3 LAMP reaction electrophoretogram; Among the figure: M:DL2000 DNA ladder; 1 negative contrast; 2～7 are respectively 10
3, 10
2, 10
1, 10
0, 10
-1, 10
-2TCID
50The nucleic acid that extracts of viral liquid be the product electrophoretogram that template is carried out the LAMP reaction;
Fig. 2 is a fluorescence LAMP collection of illustrative plates at regular time and quantity; Wherein A, B, C, D, E represent respectively to comprise 10 in the reaction
2, 10
1, 10
0, 10
-1, 10
-2The nucleic acid that the viral liquid of TCID50 extracts is the collection of illustrative plates that template is carried out fluorescence real-time quantitative LAMP reaction.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:LAMP method detects the 3 type grippe virus geneome RNA
3 type grippe virus is selected influenza virus A3/Shanghai/1/98 virus strain for use, derives from national influenza center.Observation, calculating half cell infection dosage (TCID by influenza virus pair cell infection conditions
50) method demarcate concentration, and obtain the viral liquid (10 of different concns by the method for gradient dilution
3, 10
2, 10
1, 10
0, 10
-1, 10
-2TCID
50), each concentration virus liquid is carried out RNA by following step extracts:
1. the 3 type grippe virus geneome RNA extracts:
1) adds 500 μ L RLT and 6 μ L beta-mercaptoethanols in the centrifuge tube;
2) in above-mentioned solution, add 200 μ L virus liquid, vibration mixing 1min;
3) add isopyknic 70% (v/v) ethanol mixing;
4) get 700 μ L mixed solutions and add in the interior centrifugal post of collection tube the centrifugal 15s of 12000rpm;
5) repeating step 4 once;
6) add 700 μ L RW1 damping fluids in centrifugal post, the centrifugal 15s of 12000rpm;
7) add 500 μ L RPE damping fluids in centrifugal post, the centrifugal 15s of 12000rpm;
8) repeating step 7 once;
9) liquid in the collection tube to the greatest extent, empty from once;
10) add the water dissolution RNA and the centrifugal collection of no RNA enzyme in right amount, place-70 ℃ of preservations.
2.LAMP reaction and gel electrophoresis
The LAMP reaction system is formed (primer is synthetic by Shanghai Ying Jun biotech company):
F3,5pmol; B3,5pmol; BIP, 40pmol; FIP, 40pmol; LB, 20pmol; DNTP, 1.4mM; MgSO
4, 6mM; AMV reversed transcriptive enzyme (Promega), 10U; Bst-DNApolymerase (NEB), 8U; 10 * Bst-DNA polymerase Buffer, 2.5 μ L; Viral RNA 7.5 μ L; The DEPC treating water complements to 25 μ L, mixing;
Reaction mixture is at 63 ℃ of reaction 50min, and 80 ℃ of 2min are with termination reaction then, and each reaction is got 5 μ L and carried out agarose gel electrophoresis, and observations is seen Fig. 1.
Presentation of results LAMP method can detect 3 type grippe virus RNA sensitively.
Embodiment 2: real-time fluorescence LAMP method detects the 3 type grippe virus geneome RNA
With embodiment 1 different concns virus liquid extract the RNA that obtains as follows step carry out the LAMP reaction:
The LAMP reaction system is formed (primer is synthetic by Shanghai Ying Jun biotech company):
F3,5pmol; B3,5pmol; BIP, 40pmol; FIP, 40pmol; LB, 20pmol; DNTP, 1.4mM; MgSO
4, 6mM; AMV reversed transcriptive enzyme (Promega), 10U; Bst-DNApolymerase (NEB), 8U; 10 * Bst-DNA polymerase Buffer, 2.5 μ L; The viral RNA 7.5 μ L that embodiment 1 obtains; SYBR Green I 1 μ L, the DEPC treating water complements to 25 μ L, mixing.
The LAMP reaction conditions: MJ Opticon2 instrument detects fluorescence in real time, and reaction conditions is 63 ℃ of 45s, reads plate; Carry out 40 circulations altogether.
Among Fig. 2: A, B, C, D, E represent respectively to comprise 10 in the reaction
2, 10
1, 10
0, 10
-1, 10
-2TCID
50The nucleic acid that extracts of viral liquid be the collection of illustrative plates that template is carried out fluorescence real-time quantitative LAMP reaction.
Reaction result explanation real-time fluorescence LAMP method can detect 3 type grippe virus RNA sensitively.
Embodiment 3: testing sample (patient's gargarism) LAMP reaction detection
Extract the RNA method and the reaction system of first 3 type influenza patient gargarism samples according to embodiment 1 and carry out the detection of LAMP method, reaction mixture is at 63 ℃ of reaction 50min, 80 ℃ of 2min are with termination reaction then, each reaction is got 5 μ L and is carried out agarose gel electrophoresis, observe the visible typical scalariform amplified band of gel, illustrate that the LAMP method can be used for detecting the 3 type grippe virus that the clinical case sample comprises.
Sequence table _ ST25
SEQUENCE?LISTING
＜110〉Zhejiang Center For Disease Control and Prevention
＜120〉loop-mediated isothermal amplification detection kit of 3 type grippe virus and detection method
<130>
<160>5
<170>PatentIn?version?3.4
<210>1
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>1
<210>2
<211>18
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
<210>3
<211>49
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>3
<210>4
<211>45
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>4
<210>5
<211>23
<213>Unknown
<220>
＜223〉artificial sequence
<400>5
Claims (5)
1. a 3 type grippe virus ring mediated isothermal amplification detects detection kit, and it is characterized in that: described test kit comprises following specific primer sequence:
F3:GCAGAATAAGCATCTATTGGA；
B3:GCCCCATATGTGATCCTG；
FIP:CGTATTTTGAAGTAACCCCKAGGAGGGAGACATACTTT
TGATTAACAGC；
BIP:AAGCTCAATAATGAGRTCAGATGCAGTCATTGGGAATG
CTTCCAT；
LB:GCAATTCTGAATGCATCACTCCA。
2. the loop-mediated isothermal amplification detection method of a 3 type grippe virus, described method comprises:
(1) as follows according to 3 type grippe virus hemagglutinin gene design specific primer sequence:
F3:GCAGAATAAGCATCTATTGGA；
B3:GCCCCATATGTGATCCTG；
FIP:CGTATTTTGAAGTAACCCCKAGGAGGGAGACATACTTT
TGATTAACAGC；
BIP:AAGCTCAATAATGAGRTCAGATGCAGTCATTGGGAATG
CTTCCAT；
LB:GCAATTCTGAATGCATCACTCCA；
(2) extracting testing sample RNA, is that sequence F3, B3, FIP, BIP and LB are that primer carries out the LAMP reaction in template, the step (1) with testing sample RNA;
(3) after the LAMP reaction finishes, analyze, if the LAMP reaction result is positive, then testing sample contains 3 type grippe virus.
3. the method for claim 1, it is characterized in that: described LAMP reaction system final concentration is composed as follows: F3,0.2 μ mol/L; B3,0.2 μ mol/L; BIP, 1.6 μ mol/L; FIP, 1.6 μ mol/L; LB, 0.8 μ mol/L; DNTP, 1.4mM; MgSO
4, 6mM; The AMV reversed transcriptive enzyme, 0.4U/ μ L; Bst-DNA polymerase, 0.32U/ μ L; Bst-DNA polymerase Buffer, final concentration be 1 *; Sample RNA; Solvent is a deionized water.
4. method as claimed in claim 1 or 2 is characterized in that: described LAMP reaction conditions is 60～65 ℃ of reactions 30～60min, 80 ℃ of 2min termination reactions then.
5. method as claimed in claim 3 is characterized in that: after the LAMP reaction finishes, get the LAMP reaction solution and carry out agarose gel electrophoresis, under ultra violet lamp, if trapezoid-shaped strips occurs in the EB stained gel, then the LAMP amplification is positive.
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| CN101376912B CN101376912B (en) | 2012-05-30 |
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| CN101608242A (en) * | 2009-04-09 | 2009-12-23 | 泰州亲和力生物技术有限公司 | A kind of H3 subtype flu quick-detecting type classifying method based on the RT-LAMP technology |
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| CN102559925A (en) * | 2009-06-10 | 2012-07-11 | 上海人类基因组研究中心 | Primer and method for detecting human influenza A virus H3 subtype |
| CN101591715B (en) * | 2009-07-01 | 2011-08-10 | 中国人民解放军军事医学科学院基础医学研究所 | Kit and special primer for detecting H1N1 influenza A virus and target sequence |
| CN101875933A (en) * | 2010-06-22 | 2010-11-03 | 中国人民解放军军事医学科学院微生物流行病研究所 | Kit for identifying subtypes of influenza A virus |
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