CN104962607A - Detection method for isothermal amplification of single or multiple target gene fragments - Google Patents
Detection method for isothermal amplification of single or multiple target gene fragments Download PDFInfo
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Abstract
The invention discloses a detection method for isothermal amplification of single or multiple target gene fragments. According to the method, FIB and/or BIP primer 5' end is added with a restriction enzyme cutting site sequence during LAMP amplification and is marked with fluorophore, and other parts of the primer are marked with quenching groups; after amplification, amplification results of the corresponding target genes are displayed according to a fluorescence detection, thereby accomplishing detection of single or multiple target gene fragments by one-time amplification. Compared with traditional LAMP, the method provided by the invention has the advantages of single or multiple detection, high efficiency amplification, rapid reaction, specific detection and simple operation.
Description
Technical field
The present invention relates to a kind of amplifying target genes fragment and to the method that amplification detects, belong to biology field.
Background technology
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) be a kind of new nucleic acid specificity amplification technique set up by Notomi etc., the advantage such as there is high specificity, sensitive height, simple to operate, product easily detects.This technology is widely used in molecular diagnosis field.LAMP designs 4 core primers for 6 specific regions of target sequence, utilizes the new chain synthesis of Bst archaeal dna polymerase catalysis under constant temperature with strand-displacement activity, thus makes target sequence efficient amplification.Article 4, among core primers 2 be inner primer, i.e. FIP (ForWard inner primer, FIP) and BIP (Backward inner primer, BIP).FIP comprises Flc and F2 (complementary sequence in F2c region), i.e. 5 '-Flc-F2; BIP comprises B1c (complementary sequence in B1 region) and B2, i.e. 5 '-Blc-B2.All the other two core primers are outer primer is F3 and B3.Two ring primers (Loop primes, LF and LB) are in addition added in reaction system and accelerate LAMP reaction.
LAMP reaction comprises two processes, i.e. dumbbell shaped templated synthesis stage and the cyclic amplification stage.The temperature of LAMP reaction is 60 ~ 65 DEG C, this temperature is the medium temperature of double-stranded DNA renaturation and extension, double-stranded DNA is in and partly dissociates with in the dynamic balance state of quasi integration in this temperature range, any one primer to the complementary portions of double-stranded DNA carry out base pairing extend time, another chain will dissociate, and becomes strand.Under the effect of strand displacement Bst archaeal dna polymerase, with 3 ' end of FIP primers F 2 section for starting point, match with corresponding DNA complementary sequence, start strand displacement DNA synthesis.F3 primer and F2c front end F3c complementary, with 3 ' end for starting point, by the effect of strand-displacement activity archaeal dna polymerase, first displace the DNA chain of FIP primer synthesis, synthesize self DNA simultaneously.The DNA chain that final F3 primer is synthesized into and template DNA form double-strand.But the DNA chain first synthesized by FIP primer is carried out strand displacement by F3 primer produces a strand, this strand by the F1c section of 5 ' end and F1 section complementary, there is self-base pairing, form stem ring texture.Meanwhile, BIP primer combines with the hybridization of this strand, holds as starting point with 3 ' of BIP primer, and synthesis complementary strand, loop-stem structure is opened in the process.Then, the complementary region B3c outside B3 primer and BIP primer matches and combines, and holds as starting point, under the effect of polysaccharase, synthesize new complementary strand with 3 '.By above-mentioned 2 processes, form double-stranded DNA., there is self-base pairing in the complementary region that replaced single stranded DNA exists according to two ends, forms ring texture, so the replaced DNA out of whole piece presents dumbbell structure at two ends, this structure reacts the initial structure of amplification cycles as LAMP.LAMP reacts the amplification cycles stage: first in dumbbell structure, with the Fl section of 3 ' end for starting point, with from as template, carries out DNA synthesis and extends.Meanwhile, FIP primers F 2 is hybridized with strand F2c on ring, and start new round strand replacement reaction, dissociate the double-strandednucleic acid synthesized by F1 section.Equally, the single-chain nucleic acid dissociateed also can form loop-stem structure.Loop-stem structure exists single stranded form B2c, and the B2 on BIP primer is hybrid with it, and starts new round amplification, through identical process, forms loop-stem structure again.By this process, result complementary sequence on same chain goes round and begins again and forms structure not of uniform size.LAMP reacts the target DNA that can increase special, efficiently, fast, makes the amount of product reach 10 within an hour
9individual copy.
After LAMP amplification, the detection of its product can be observed by agarose electrophoresis poststaining.Comparatively easy method directly adds SYBR Green I dyeing in the product, and presenting green is positive reaction, and orange red is negative reaction.Also can be judged by the turbidity of amplification by product magnesium pyrophosphate precipitation, liquid is muddy, and what centrifugal or adularescent precipitated is positive reaction, and without this phenomenon is then negative reaction.More simple method adds visible dyes in the reactive mixture now, and the color of positive reaction pipe becomes green from light gray, and negative reaction pipe then keeps original light gray.But whether these methods all can only detect LAMP reaction and carry out, and can not identify the specific amplification for particular target sequence, cause LAMP when testing goal sequence, and the judgement of its result lacks specificity.Therefore, traditional LAMP detects and is difficult to realize detecting while multipurpose fragment, this strongly limits the widespread use of LAMP.
In view of the above problems, some researchs are devoted to develop multiple LAMP detection technique.Realizing multiple LAMP, to detect modal method be find restriction enzyme digestion sites in target sequence, LAMP product is used digestion with restriction enzyme, by the postdigestive LAMP product of electrophoresis, corresponding with corresponding target sequence according to different electrophoretic band sizes.But the method needs two steps to complete, during the LAMP product of restriction fragment different sizes, longer and enzyme consuming time is cut not exclusively, several the electrophoretic bands that caused a target sequence usually corresponding, and the result difficulty of multiple LAMP is judged.The new technology that another kind realizes multiple LAMP detection is combined LAMP amplified reaction and Manganic pyrophosphate complex initiation.But the multiple LAMP detection technique that the method and restriction enzyme mediate is the same, all needs two steps to complete, is first LAMP amplification, then corresponds to corresponding target sequence by Manganic pyrophosphate complex initiation.The method complex operation, need specific test kit to carry out purifying to LAMP product, sequencing procedure needs special personnel, and the sequenator that cannot bear of common lab and sequencing reagent.These inferior positions limit promoting the use of of the method.In addition, existing multiple LAMP detection technique all cannot realize rapid detection, completes multiple LAMP and detects and be consuming timely greater than 2.5 hours.Because the sensitivity of LAMP reaction is high, the operation of uncapping carrying out LAMP product exists later LAMP experiment pollutes greatly.
To sum up, no matter LAMP of the prior art also exists the technical problem being difficult to overcome in single amplification or the detection of multiplex amplification.
Summary of the invention
The object of the invention is the principle improving LAMP reaction, based on LAMP reaction, in conjunction with digestion with restriction enzyme and fluoroscopic examination principle, set up a kind of easy and simple to handle, single or multiple LAMP detection of high specificity, highly sensitive, that detection speed is fast single stage method, to realize widely using of LAMP technology.
Based on above-mentioned purpose, the present invention provide firstly a kind of amplifying target genes fragment and to the method that amplification detects, said method comprising the steps of:
(1) from 3 ' end of described goal gene fragment, set the first arbitrary sequence F1c, the second arbitrary sequence F2c and the 3rd arbitrary sequence F3c, from 5 ' end of described goal gene fragment, set the 4th arbitrary sequence B1, the 5th arbitrary sequence B2 and the 6th arbitrary sequence B3;
(2) provide primer EFIP, described primer EFIP contains the sequence F2 with sequence F2c complementation, is connected with the sequence identical with sequence F1c at the 5 ' end of described sequence F2, is connected with one section of restriction enzyme identification fragment at described sequence F1c 5 ' end; There is provided primer EBIP, described primer EBIP contains the sequence identical with sequence B 2, is connected with the sequence B 1c with sequence B 1 complementation at 5 ' end of described sequence B 2, is connected with one section of restriction enzyme identification fragment at described sequence B 1c 5 ' end; Wherein be marked with a fluorophor at the 5 ' end of primer EFIP and/or primer EBIP, have a quenching of fluorescence group at described other position mark be labeled in the primer of fluorophor except described restriction enzyme enzyme fragment, described quenching of fluorescence group can fluorophor described in quencher;
(3) under chain shift-type polysaccharase, melting temperature(Tm) conditioning agent, primer, described restriction enzyme and damping fluid exist, application target gene fragment is as template amplification DNA;
(4) amplification of detecting step (3).
Preferably, be detected as visible color change detection, electrophoresis detection or real-time fluorescence described in step (4) detect.
Step (3) described chain shift-type polysaccharase can be Bst archaeal dna polymerase, described melting temperature(Tm) conditioning agent can be trimethyl-glycine.
In a preferred technical scheme, institute provides primer also to comprise containing the primers F 3 with sequence F3c complementation in described step (2), and contains the primer B3 identical with sequence B 3.
Preferably, institute provides primer also to comprise to contain pair of primers LF and LB of complementary between F1c-F2c and between B1c-B2c in described step (2).
Amplification described in step (2) can be carried out in 60 ~ 66 DEG C, and in a preferred technical scheme, described in step (2), amplification carries out in 63 ~ 65 DEG C.
In a preferred technical scheme, described primer sequence is selected from one sequence combination:
(1) sequence of described primer EFIP is by shown in SEQ ID NO:4, the sequence of described primer EBIP is by shown in SEQ ID NO:5, the sequence of described primers F 3 is by shown in SEQ ID NO:1, the sequence of described primer B3 is by shown in SEQ ID NO:2, the sequence of described primer LF is by shown in SEQ IDNO:6, and the sequence of described primer LB is by shown in SEQ ID NO:7; Or
(2) sequence of described primer EFIP is by shown in SEQ ID NO:11, the sequence of described primer EBIP is by shown in SEQ ID NO:12, the sequence of described primers F 3 is by shown in SEQ ID NO:8, the sequence of described primer B3 is by shown in SEQ ID NO:9, the sequence of described primer LF is by shown in SEQ IDNO:13, and the sequence of described primer LB is by shown in SEQ ID NO:14.
In above-mentioned combined sequence, (1) group combination is used for the detection of Listeria monocytogenes bacterium, and (2) group combination is used for the detection of Listeria ivanovii bacterium.
Preferably, the fluorophor that in described combined sequence, primer EFIP sequence SEQ ID NO:4 marks is FAM, and the fluorophor that primer EFIP sequence SEQ ID NO:11 marks is HEX.
In another preferred technical scheme of the present invention, the goal gene fragment that the inventive method can use one or more above step (2) described primers once to increase corresponding also detects, wherein, the fluorophor that the primer matched from each goal gene fragment marks is different, and demonstrates the result of otherness in the detection of step (4).
Preferably, described primer sequence is one sequence combination:
(1) sequence of described primer EFIP is by shown in SEQ ID NO:4, the sequence of described primer EBIP is by shown in SEQ ID NO:5, the sequence of described primers F 3 is by shown in SEQ ID NO:1, the sequence of described primer B3 is by shown in SEQ ID NO:2, the sequence of described primer LF is by shown in SEQ IDNO:6, and the sequence of described primer LB is by shown in SEQ ID NO:7; And
(2) sequence of described primer EFIP is by shown in SEQ ID NO:11, the sequence of described primer EBIP is by shown in SEQ ID NO:12, the sequence of described primers F 3 is by shown in SEQ ID NO:8, the sequence of described primer B3 is by shown in SEQ ID NO:9, the sequence of described primer LF is by shown in SEQ IDNO:13, and the sequence of described primer LB is by shown in SEQ ID NO:14.
In above-mentioned combined sequence, (1) group combination is used for the detection of Listeria monocytogenes bacterium, (2) group combination is used for the detection of Listeria ivanovii bacterium, the fluorophor that wherein ID of primer EFIP sequence SEQ described in two kinds of combined sequence NO:4 marks is FAM, and the fluorophor that primer EFIP sequence SEQ ID NO:11 marks is HEX.
On the other hand, present invention also offers a kind of test kit detected for amplifying target genes fragment and to amplification, described test kit comprises:
(1) from 3 ' end of described goal gene fragment, set the first arbitrary sequence F1c, second arbitrary sequence F2c and the 3rd arbitrary sequence F3c, from 5 ' end of described goal gene fragment, set the 4th arbitrary sequence B1, during the 5th arbitrary sequence B2 and the 6th arbitrary sequence B3, primer EFIP is provided, described primer EFIP contains the sequence F2 with sequence F2c complementation, be connected with the sequence identical with sequence F1c at the 5 ' end of described sequence F2, be connected with one section of restriction enzyme identification fragment at described sequence F1c 5 ' end; There is provided primer EBIP, described primer EBIP contains sequence B 2, is connected with the sequence B 1c with sequence B 1 complementation at 5 ' end of described sequence B 2, is connected with one section of restriction enzyme identification fragment at described sequence B 1c 5 ' end; Wherein be marked with a fluorophor at the 5 ' end of primer EFIP and/or primer EBIP, have a quenching of fluorescence group at described other position mark be labeled in the primer of fluorophor except described restriction enzyme enzyme fragment, described quenching of fluorescence group can fluorophor described in quencher;
(2) chain shift-type polysaccharase, melting temperature(Tm) conditioning agent, primer, described restriction enzyme, damping fluid.
Preferably, described test kit also comprise containing with the primers F 3 of sequence F3c complementation, with containing the primer B3 identical with sequence B 3.
Preferably, described test kit also comprises pair of primers LF and LB containing complementary between F1c-F2c and between B1c-B2c.
Preferably, described chain shift-type polysaccharase is Bst polysaccharase, described melting temperature(Tm) conditioning agent is trimethyl-glycine.
The present invention is on the basis of original LAMP primer, core primers FIP and/or BIP of LAMP is improved, at 5 ' end interpolation one section of cleavage sequence (called after Es) of FIP and/or BIP, this sequence can the specific identification of being limited property restriction endonuclease, and the primer after improvement is called EFIP and EBIP.It should be noted that, in order to the consistence stated, for the above-mentioned core primers of reaction system in the present invention, no matter whether be added cleavage sequence, be EFIP and EBIP by Uniform Name in the claims without exception.In specific embodiment of the invention scheme, how FIP and BIP is specifically being added in the mode of cleavage sequence, should be as the criterion with the concrete disclosed content of this embodiment, instead of only depend on the name title of this primer.When synthesizing EFIP and the EBIP primer for different target sequence, at the fluorophor that 5 ' the end mark of EFIP and/or EBIP is different, the central marker quencher of primer, this quencher quencher 5 ' can hold fluorophor.
In reaction system of the present invention, EFIP and/or EBIP is used to substituted for original FIP and BIP, this reaction is carried out under constant temperature, its reaction process and LAMP reacting phase are seemingly, along with the carrying out of reaction, the complementary strand CEs taking Es as template has been synthesized in reaction mixture, this double-strand can specific identification, cutting by the restriction enzyme in reaction system, fluorophor and quencher separate by this process, the fluorescent signal of release can be detected by fluorimetric detector, and different fluorescent signals represents different target sequences.Meanwhile, after the Es sequence of EFIP/ or EBIP is sheared, this primer becomes core primers FIP and BIP of LAMP reaction, LAMP is reacted and proceeds (see Fig. 1).
The advantage applies of method provided by the invention (called after MERT-LAMP technology) in:
1. compare with LAMP
More common LAMP technology, when the MERT-LAMP technology that the present invention sets up carries out unique sequence detection, its result interpretation, except carrying out interpretation by conventional LAMP result read method, can also use real-time fluorescence Cleaning Principle to carry out interpretation, its detection rates is faster, operates simpler.
Advantage of the present invention is also to carry out multisequencing detection in same reaction system, has promoted the application of LAMP technology.When using MERT-LAMP to carry out multisequencing detection, consume shorter detection time than LAMP, and detection sensitivity and LAMP reacting phase are together.
2. compare with existing multiple LAMP technology
More existing multiple LAMP technology, the present invention can in same reaction tubes a step, complete multisequencing fast and detect, do not need electrophoresis or order-checking LAMP product, avoid opening LAMP reaction tubes, decrease the pollution to subsequent operations.The result of MERT-LAMP technology reads simple, only to the interpretation of fluorescent signal, can need detect in real time.MERT-LAMP technology presents positive findings, the shortlyest consuming timely only needs 12 minutes, the longest 30 minutes consuming time, time less than existing multiple LAMP technology fast 2.5 to 3.In addition, the test kit that MERT-LAMP technology uses is identical with common LAMP, does not need high sequencing reagent.
3. with qPCR Technical comparing
Comparatively qPCR technology, the present invention has stronger Multiple detection ability, can to EFIP with EBIP with the different luminophore of tense marker.Based on the MERT-LAMP technology of LAMP reaction, detection sensitivity is obviously better than qPCR technology, and Monitoring lower-cut 10 times is sensitive in qPCR technology.In detection rates, MERT-LAMP technology is better than qPCR technology equally, MERT-LAMP technology presents positive findings, the shortlyest consuming timely only need 12 minutes, about 20 minutes are shortened than qPCR, when to detect the DNA of respective Monitoring lower-cut level simultaneously, MERT-LAMP technology needs 30 minutes, and qPCR technology then needs 53 minutes.Therefore, the present invention no matter in Multiple detection ability, detection rates, or is better than qPCR technology in detectability.
Accompanying drawing explanation
Fig. 1 .MERT-LAMP method Cleaning Principle schematic diagram;
Fig. 2 .MERT-LAMP design of primers schematic diagram;
Fig. 3 .MERT-LAMP amplification detects schematic diagram;
The real-time Turbidity measurement schematic diagram of Fig. 4 .MERT-LAMP amplification;
Fig. 5. differing temps MERT-LAMP amplification real-time fluorescence detects schematic diagram;
Fig. 6. substance MERT-LAMP amplification real-time fluorescence detects schematic diagram;
Fig. 7. multiple MERT-LAMP amplification real-time fluorescence detects schematic diagram;
Fig. 8. multiple MERT-LAMP method Evaluation on specificity figure.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to protection scope of the present invention.
In order to verify the feasibility of method provided by the invention (MERT-LAMP technology), two pathogenic Liszt's bacterial classifications, Listeria monocytogenes and listeria ivanovii are selected as model bacterium.According to Listeria monocytogenes and listeria ivanovii species-specific genes lmo0733 (GenBank GeneID:985919, Listeria monocytogenes, and smcL (GenBank Gene ID:11161600 LM), Listeria ivanovii, Liv) two cover MERT-LAMP primers are designed, lmo0733-MERT-LAMP primer is for detecting Listeria monocytogenes, and smcL-MERT-LAMP primer is for detecting listeria ivanovii.Lmo0733 is present in all serotype of single increasing Liszt, smcL is present in all hypotypes of listeria ivanovii, the specificity of goal gene is good, can by Listeria monocytogenes, Yi Shi Liszt and other closely close bacterial classifications (as listeria grayi, Weir listeria bacteria, Ying Nuoke listeria bacteria) and Pseudomonas (as bacillus) distinguish.LAMP primer design software PrimerExplorer V4 (EikenChemical) (http://primerexplorer.jp/e/) and primer-design software Primer Premier 5.0 is utilized to design MERT-LAMP primer, and the Auele Specific Primer obtained is carried out sequence alignment analysis in ncbi database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), mate to get rid of non-specific that primer and other species sequence may exist, finally obtain the special MERT-LAMP amplimer of after optimizing two covers.Fig. 2 (2-A is the MERT-LAMP primer for Listeria monocytogenes amplification, and 2-B is the MERT-LAMP primer for listeria ivanovii amplification) is seen in the position of design of primers and direction, and sequence is in table 1.
Table 1 primer used in this application
(1) LM means L.monocytogenes: Listeria monocytogenes
(2) this primer does not add cleavage sequence, and adding this primer is reaction in order to accelerate MERT-LAMP
(3) this primer with the addition of cleavage sequence, and marked fluorophor, and the sequence after mark is:
5′-FAM-TGCAATG-CGGTGCT(BHQ-1)AATCTGACAAACAACCAT-TATAGTGATCCTGAAACTAGCGTT-3′
FAM refers to 6-Fluoresceincarboxylic acid, and BHQ-1 (Blackhole quencher1) refers to fluorescence quencher 1
(4) this primer does not add cleavage sequence, and adding this primer is reaction in order to accelerate MERT-LAMP
(5) LI means L.ivanovii: listeria ivanovii
(6) this primer with the addition of cleavage sequence, and marked fluorophor, and the sequence after mark is:
5′-HEX-TGCAATG-ACGGGTGTTT(BHQ-1)GATGAGGATACATTT-GTCGTCATTTTAAACGAAGCCTT-3′
HEX refers to chlordene-6-methyl fluorescein
(7) F, upstream primer
(8) R, downstream primer
(9) sequence after mark is:
5′-FAM-TGTGTGCCGTTATAGCAATAATTTCTGCG-BHQ-1-3
(10) sequence after mark is:
5′-HEX-CAGCCACTCCACCATCTTCCAAAGCA-BHQ-1-3′
SEQ ID NO:15 to SEQ ID NO:26 is the common LAMP primer of two covers for Listeria monocytogenes and listeria ivanovii, for comparing the susceptibility of LAMP and MERT-LAMP, SEQ IDNO:27 to SEQ ID NO:32 is the two cover Real-time primers for Listeria monocytogenes and listeria ivanovii, for comparing the susceptibility of qPCR and MERT-LAMP.
The reagent using and relate in the present invention:
Loopamp Kit (Eiken Chemical Co.Ltd., Tokyo, Japan) is purchased from Japanese Rong Yan company.DNA extraction kit (QIAamp DNA minikits; Qiagen, Hilden, Germany) be purchased from German Qiagen company.QPCR reaction system mixture Premix (Takara Bio, Inc., Otsu, Japan, dNTP and damping fluid) is purchased from Takara bio tech ltd, Beijing.DL50DNAMarker and DL50DNA Marker is purchased from precious biotechnology (Dalian) company limited.All the other reagent are commercially available somatotype straight product.
The key instrument using and relate in the present invention's experiment: the real-time turbidimeter LA-320C of Loopamp (Eiken Chemical Co., Ltd, Japan) is purchased from Japanese Rong Yan company.Real-time fluorescence detector is Rotor-Gene Q Real-Time System (Qiagen), German Qiagen product; Electrophoresis equipment is Jun Yi east, Beijing electrophoresis equipment company limited product; Gel imaging system is Bio-RadGel Dox XR, U.S. Bio-Rad product.
The extraction of the genomic dna of Listeria monocytogenes, listeria ivanovii and other kind bacterium:
Genome extracts: the extraction of bacterial genomes uses DNA extraction kit (the QIAamp DNA minikits of Qiagen company; Qiagen, Hilden, Germany), operate to specifications.Utilize ultraviolet spectrophotometer to measure the concentration of genomic dna and purity, Listeria monocytogenes and listeria ivanovii genomic dna are with GE buffer serial-dilution (from 25ng, 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg to 62.5fg).The all a small amount of packing of various genomic dna ,-20 DEG C save backup.
The single amplification of embodiment 1.MERT-LAMP:
(1) the single amplification of MERT-LAMP (i.e. standard reaction) system is as follows: the concentration of primer EFIP and FIP is respectively 20pmol, the concentration of primer BIP is 40pmol, the concentration of primers F 3 and B3 is 5pmol, the concentration of primer LF and BF is 20pmol, the MgSO of the Betain of 10mM, 6mM
4, 10 × Bst DNA polymerase buffer liquid of the dNTP of 1mM, 2.5 μ l, the strand displacement archaeal dna polymerase of 8U, the DNA restriction enzyme of 15U, the template of 1 μ l, adds deionized water to 25 μ l.Whole reaction is carried out in fluorescence detector (Rotor-Gene Q Real Time System, Qiagen), isothermal duplication 63 ~ 65 DEG C of 1h, 80 DEG C of 5min termination reactions.
(2) feasibility checking:
Under standards system condition, add MERT-LAMP primer and corresponding DNA profiling designed by lmo0733 and smcL specific gene, lmo0733-MERT-LAMP reaction is for the Listeria monocytogenes that increases (Fig. 3-A1,2,3-B1,2), smcl-MERT-LAMP reaction is for the listeria ivanovii that increases (Fig. 3-A3,4,3-B3,4), MERT-LAMP technical feasibility is confirmed by visible color method of changing and electrophoresis assays.
Visible color method of changing: MERT-LAMP produces a large amount of pyrophosphate ions while synthetic DNA, and this ion can capture the mn ion be combined with fluorexon, makes fluorexon recover unbound state and fluoresce.The magnesium ion that this light-emitting admixture can produce in reaction is combined, and fluorescence is enhanced.Can pass through fluorescence visual detection colour-change sentence read result, positive reaction pipe becomes green from light gray, and negative reaction keeps light grey constant, sees Fig. 3 A.Fig. 3-A1 is the positive findings of lmo0733-MERT-LAMP primer amplification Listeria monocytogenes, and Fig. 3-A2 is the negative findings of lmo0733-MERT-LAMP reaction; Fig. 3-A3 is the positive findings of smcL-MERT-LAMP primer amplification listeria ivanovii, and Fig. 3-A4 is the negative findings of smcL-MERT-LAMP reaction.Proved by visual detection result, the MERT-LAMP designed by the present invention is feasible, can carry out augmentation detection to target sequence.In addition, visual detection result also demonstrates two covers of the present invention designed by MERT-LAMP principle and can be used for object bacterium for the MERT-LAMP primers of Listeria monocytogenes and listeria ivanovii and detect.
Electrophoresis assays: due to MERT-LAMP reaction with LAMP reacting phase seemingly, its product is also the loop-stem structure of target sequence formation and the DNA fragmentation mixture of many rings Cauliflower spline structure of a series of inverted repeat, after electrophoresis, positive amplification manifests the staged collection of illustrative plates of different size zone composition on gel, there is not amplified band in negative findings, sees Fig. 3 B.Fig. 3-B1 is the positive findings of lmo0733-MERT-LAMP primer amplification Listeria monocytogenes, and Fig. 3-B2 is the negative findings of lmo0733-MERT-LAMP reaction; Fig. 3-B3 is the positive findings of smcL-MERT-LAMP primer amplification listeria ivanovii, and Fig. 3-B4 is the negative findings of smcL-MERT-LAMP reaction.Proved that by electrophoresis detection result the MERT-LAMP technical feasibility designed by the present invention can carry out augmentation detection to target sequence further.In addition, electrophoresis detection result also demonstrates two covers of the present invention designed by MERT-LAMP principle and can be used for object bacterium for the MERT-LAMP primers of Listeria monocytogenes and listeria ivanovii and detect.
Real-time fluorescence detection method:
Under standards system condition, add MERT-LAMP primer and corresponding DNA profiling designed by lmo0733 and smcL specific gene, the reaction of lmo0733-MERT-LAMP real-time amplification is used for the detection to object bacterium Listeria monocytogenes, and the reaction of smcL-MERT-LAMP real-time amplification is used for the detection to object bacterium listeria ivanovii.Under standards system condition, according to MERT-LAMP reaction principle and designed primer, the interpretation of result relies on the different fluorescent signals that enzyme cuts rear generation, is received obtain stable fluorescence curve (Fig. 4) by fluorimetric detector.Fig. 4 A is lmo0733-MERT-LAMP primer (FAM mark) real-time amplification detected result, and Fig. 4-A1 is the positive findings detecting Listeria monocytogenes, and amplified signal is greater than threshold value, and Fig. 4-A2 is negative control result, does not produce any amplification curve.Fig. 4 B is smcL-MERT-LAMP primer (HEX mark) real-time amplification detected result, and Fig. 4-B1 is the positive findings detecting listeria ivanovii, and amplified signal is greater than threshold value, and Fig. 4-B2 is negative control result, does not produce any amplification curve.Proved by real-time fluorescence detected result, MERT-LAMP result can carry out interpretation by real-time fluorescence, thus provide foundation for the interpretation of Multiple detection result, secondly fluoroscopic examination result further demonstrates the MERT-LAMP technical feasibility designed by the present invention, can carry out augmentation detection to target sequence.In addition, real-time fluorescence detected result also demonstrates two covers of the present invention designed by MERT-LAMP principle and can be used for object bacterium for the MERT-LAMP primers of Listeria monocytogenes and listeria ivanovii and detect.
(3) optimal reaction temperature of MERT-LAMP technology is measured
Under standards system condition, add the DNA profiling of listeria ivanovii MERT-LAMP primer and listeria ivanovii, its template concentrations is 2.5pg/ μ l.Reaction is carried out (61 ~ 66 DEG C) under constant temperature, and application of results real-time fluorescence detector detects, and obtains different dynamic curve diagrams at different temperature, sees Fig. 5.Fig. 5 A to 5F is respectively the amplification of MERT-LAMP amplified reaction under 61 ~ 66 DEG C of conditions, under 63 ~ 65 DEG C of amplification conditions, MERT-LAMP amplification curve is stablized, and occurs the time of positive findings early, therefore 63 ~ 65 DEG C of recommended optimal reaction temperatures as MERT-LAMP technology.Follow-up study of the present invention is selected to carry out under 64 DEG C of conditions.
(4) MERT-LAMP detects the sensitivity of single sample
Under standards system condition, serial dilution Listeria monocytogenes and listeria ivanovii template DAN (2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg and 62.5fg/ microlitre), each concentration template of 1 microlitre adds in reaction system, is detected by real-time fluorescence, obtain stable fluoroscopic examination figure, see Fig. 6.6A is lmo0733-MERT-LAMP primer (FAM mark) real-time amplification detected result, 6-A1 to 6-A5 amplified signal curve is greater than threshold value, interpretation is the positive findings detecting Listeria monocytogenes, corresponding Listeria monocytogenes template concentrations is (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 6-A6 to 6-A8, amplified signal curve is less than threshold value, for detecting negative findings, corresponding Listeria monocytogenes template concentrations is 125fg, 62.5fg/ microlitre and negative control tube, therefore the Monitoring lower-cut of MERT-LAMP technology for detection Listeria monocytogenes nucleic acid is 250fgDNA/ reaction tubes.6B is smcL-MERT-LAMP primer (HEX mark) real-time amplification detected result, 6-B1 to 6-B5 amplified signal curve is greater than threshold value, interpretation is the positive findings detecting listeria ivanovii, corresponding listeria ivanovii template concentrations is (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 6-A6 to 6-A8, amplified signal curve is less than threshold value, for detecting negative findings, corresponding listeria ivanovii template concentrations is 125fg, 62.5fg/ microlitre and negative control tube, therefore the Monitoring lower-cut of MERT-LAMP technology for detection listeria ivanovii nucleic acid is 250fg DNA/ reaction tubes.
According to the result that Fig. 6 presents, MERT-LAMP technology is 250fg DNA/ reaction tubes for the Monitoring lower-cut of single target sequence, and the shortest detection time consumed is approximately 12 minutes, and the DNA detecting lowest detectable limit level also only consumes about 30 minutes.When in reaction system, genomic templates amount is reduced to below 250fg, there is not positive amplification in MERT-LAMP reaction.According to the result of interpretation, the remolding sensitivity qPCR method of MERT-LAMP amplified reaction is high 10 times, in table 2.
Embodiment 2.MERT-LAMP multiplex amplification
(1) MERT-LAMP multiple reaction system is as follows: the concentration of primer EFIP and FIP is respectively 20pmol, the concentration of primer BIP is 40pmol, and the concentration of primers F 3 and B3 is 10pmol, and the concentration of primer LF and BF is 10pmol, the MgSO of the Betain of 10mM, 6mM
4, 10 × Bst DNA polymerase buffer liquid of the dNTP of 1mM, 2.5 μ l, the strand displacement archaeal dna polymerase of 8U, the DNA restriction enzyme of 15U, the template of 1 μ l, adds deionized water to 25 μ l.Whole reaction is carried out in fluorescence detector (Rotor-Gene Q Real Time System, Qiagen), isothermal duplication at 64 DEG C of 1h, 80 DEG C of 5min termination reactions.
(2) Multiple detection ability and the sensitivity of MERT-LAMP is verified
In order to the multiplex amplification realizing MERT-LAMP technology detects, the present invention is optimized to adapt to Multiple detection to standards system, sets up Multiple detection system.Under Multiple detection system, add the template of two cover primers and correspondence in multiple reaction mixture simultaneously, detected by fluorescence detection equipment, obtain stable multi-fluorescence detection figure, see Fig. 7, 7A and 7B real-time amplification curve is produced simultaneously, the amplification curve of 7A comes from FAM sense channel, represent lmo0733-MERT-LAMP primer (FAM mark) real-time amplification detected result, 7-A1 to 7-A5 amplified signal curve is greater than threshold value, interpretation is the positive findings detecting Listeria monocytogenes, corresponding Listeria monocytogenes template concentrations is (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 7-A6 to 7-A8, amplified signal curve is less than threshold value, for detecting negative findings, corresponding Listeria monocytogenes template concentrations is 125fg, 62.5fg/ microlitre and negative control tube, therefore, when using MERT-LAMP technology to carry out Multiple detection, be 250fg DNA/ reaction tubes to the Monitoring lower-cut of Listeria monocytogenes nucleic acid.The amplification curve of 7B comes from HEX sense channel, represent smcL-MERT-LAMP primer (HEX mark) real-time amplification detected result, 7-B1 to 7-B5 amplified signal curve is greater than threshold value, interpretation is the positive findings detecting listeria ivanovii, corresponding listeria ivanovii template concentrations is (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 7-B6 to 7-B8, amplified signal curve is less than threshold value, for detecting negative findings, corresponding listeria ivanovii template concentrations is 125fg, 62.5fg/ microlitre and negative control tube, therefore, when using MERT-LAMP technology to carry out Multiple detection, be 250fg DNA/ reaction tubes to the Monitoring lower-cut of listeria ivanovii nucleic acid.From the result of Fig. 7, MERT-LAMP reaction system can carry out the detection of two target sequences simultaneously, demonstrates the Multiple detection ability of MERT-LAMP technology, can by the detection of MERT-LAMP technology popularization to many aim sequences.MERT-LAMP is when detecting multiple target sequence in application, and its detection time and detection sensitivity detect with simple sequence the change not having matter, and the shortest detection time is about 12 minutes, and two sensitivity of overlapping templates are 250fg DNA/ reaction tubes, in table 2.
Table 2 is applied the detection of different amplification techniques to listeria ivanovii and is compared
(3) Evaluation on specificity:
With the genomic dna (Listeria monocytogenes of common pathogenic bacterium and conditioned pathogen, listeria ivanovii, Ying Nuoke listeria bacteria, Weir listeria bacteria, Xi Er listeria bacteria, listeria grayi, Bacillus cereus, enteropathogenic Escherichia coli, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) evaluate the specificity of MERT-LAMP reaction system as template, in table 3.Two cover MERT-LAMP primer (lmo0733-MERT-LAMP primers, smcL-MERT-LAMP primer) added in MERT-LAMP multiplex amplification system simultaneously, then augmentation detection is carried out, the genomic DNA template that amplified reaction pipe 1 to 12 adds is the Listeria monocytogenes (1/2a of 12 serotypes, 3a, 1/2b, 3b, 7, 1/2c, 3c, 4a, 4c, 4b, 4d and 4e), reaction tubes 13, the genomic DNA template of 14 is listeria ivanovii reference culture and listeria ivanovii isolated strains, reaction tubes 15 to 35 is the genomic DNA template of other bacteriums, reaction tubes 36 is negative control tube, specificity identification the results are shown in Figure 8.8A and 8B real-time amplification curve is produced simultaneously, the amplification curve of 8A comes from FAM sense channel, represent lmo0733-MERT-LAMP primer (FAM mark) real-time amplification detected result, 8-A1 to 8-A12 amplified signal curve is greater than threshold value, interpretation is the positive findings detecting Listeria monocytogenes, corresponding genomic DNA template is different serotypes Listeria monocytogenes (1/2a, 3a, 1/2b, 3b, 7, 1/2c, 3c, 4a, 4c, 4b, 4d and 4e), 8-A13 to 8-A36, amplified signal curve is less than threshold value, for detecting negative findings, corresponding genomic DNA template is listeria ivanovii, other bacteriums and negative control pipe, the amplification curve of 8B comes from HEX sense channel, represent smcL-MERT-LAMP primer (HEX mark) real-time amplification detected result, 8-B13 to 8-B14 amplified signal curve is greater than threshold value, interpretation is the positive findings detecting listeria ivanovii, and corresponding genomic DNA template is listeria ivanovii reference culture and listeria ivanovii isolated strains, 8-B1 to 8-B12,8-B15 to 8-B36 amplified signal curve is less than threshold value, and interpretation is negative result, the genomic DNA template that 8-B1 to 8-B12 is corresponding is different serotypes Listeria monocytogenes (1/2a, 3a, 1/2b, 3b, 7,1/2c, 3c, 4a, 4c, 4b, 4d and 4e), 8-B15 to 8-B36, corresponding genomic DNA template is other bacteriums and negative control pipe, therefore, MERT-LAMP technology can accurately differentiate Listeria monocytogenes and listeria ivanovii, illustrates that the specificity of MERT-LAMP method is good.
Table 3 bacterial strain uses therefor of the present invention
Note:
*u, does not identify bacterial strain;
aTCC, American Type Culture Collecti; NCTC, Britain's Type Culture Collection; ICDC, Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an.
Comparative examples: Real-time RCR method reaction system
Wherein, the upstream primer sequence detected for Listeria monocytogenes is SEQ ID NO:27, downstream primer sequence is SEQ ID NO:28, probe sequence is SEQ ID NO:29, the upstream primer sequence detected for listeria ivanovii is SEQ ID NO:30, downstream primer sequence is SEQ IDNO:31, and probe sequence is SEQ ID NO:32.
Amplification also measurement result on real-time fluorescence quantitative PCR instrument:
At fluorescence detector (Rotor-Gene Q Real Time System, Qiagen) Real-time reaction is carried out in, after amplification terminates, get same analysis of threshold data after deduction background fluorescence signal, determine Ct (cycle threshold) value of this reaction.
Common LAMP reaction system:
The reaction system of LAMP: cumulative volume is 25 μ l, comprises 40pmol inner primer FIP and BIP, 5pmol outer primer F3 and B3,20pmol ring primer LF and BF, 20mM Tris-HCl (pH 8.8), 10mM KCl, 4mM MgSO
4, 10mM (NH
4)
2sO
4, 0.1%Tween 20,0.8M trimethyl-glycine, 1.4mM dNTPs, 1 μ l Bst archaeal dna polymerase (8U/mL), 1 μ l FD reagent (bright general fluorescent visual reagent) and 1 μ l DNA profiling.The reaction conditions of LAMP is all constant temperature 64 DEG C, and the reaction times is 60min.80 DEG C of 5min termination reactions.
Wherein, the FIP detected for Listeria monocytogenes be SEQ ID NO:17, BIP is SEQ IDNO:18, F3 be SEQ ID NO:15, B3 be SEQ ID NO:16, LF be SEQ ID NO:19, LB is SEQ ID NO:20.The FIP detected for listeria ivanovii be SEQ ID NO:23, BIP is SEQ ID NO:24, F3 be SEQ ID NO:21, B3 be SEQ ID NO:22, LF be SEQ ID NO:25, LB is SEQ ID NO:26.
Claims (15)
1. amplifying target genes fragment to the method that amplification detects, said method comprising the steps of:
(1) from 3 ' end of described goal gene fragment, set the first arbitrary sequence F1c, the second arbitrary sequence F2c and the 3rd arbitrary sequence F3c, from 5 ' end of described goal gene fragment, set the 4th arbitrary sequence B1, the 5th arbitrary sequence B2 and the 6th arbitrary sequence B3;
(2) provide primer EFIP, described primer EFIP contains the sequence F2 with sequence F2c complementation, is connected with the sequence identical with sequence F1c at the 5 ' end of described sequence F2, is connected with one section of restriction enzyme identification fragment at described sequence F1c 5 ' end; There is provided primer EBIP, described primer EBIP contains sequence B 2, is connected with the sequence B 1c with sequence B 1 complementation at 5 ' end of described sequence B 2, is connected with one section of restriction enzyme identification fragment at described sequence B 1c 5 ' end; Wherein be marked with a fluorophor at the 5 ' end of primer EFIP and/or primer EBIP, have a quenching of fluorescence group at described other position mark be labeled in the primer of fluorophor except described restriction enzyme enzyme fragment, described quenching of fluorescence group can fluorophor described in quencher;
(3) under chain shift-type polysaccharase, melting temperature(Tm) conditioning agent, primer, described restriction enzyme and damping fluid exist, application target gene fragment is as template amplification DNA;
(4) amplification of detecting step (3).
2. method according to claim 1, is characterized in that, the be detected as detection of visible color method of changing, electrophoresis detection or real-time fluorescence described in step (4) detect.
3. method according to claim 1, is characterized in that, described chain shift-type polysaccharase is Bst archaeal dna polymerase, described melting temperature(Tm) conditioning agent is trimethyl-glycine.
4. method according to claim 1, is characterized in that, institute provides primer also to comprise containing the primers F 3 with sequence F3c complementation in described step (2), and contains the primer B3 identical with sequence B 3.
5. method according to claim 1, is characterized in that, institute provides primer also to comprise to contain pair of primers LF and LB of complementary between F1c-F2c and between B1c-B2c in described step (2).
6. the method according to claim arbitrary in Claims 1 to 5, is characterized in that, described in step (2), amplification carries out in 60 ~ 66 DEG C.
7. method according to claim 6, is characterized in that, described in step (2), amplification carries out in 63 ~ 65 DEG C.
8. method according to claim 7, is characterized in that, described primer sequence is selected from one sequence combination:
(1) sequence of described primer EFIP is by shown in SEQ ID NO:4, the sequence of described primer EBIP is by shown in SEQ ID NO:5, the sequence of described primers F 3 is by shown in SEQ ID NO:1, the sequence of described primer B3 is by shown in SEQ ID NO:2, the sequence of described primer LF is by shown in SEQ IDNO:6, and the sequence of described primer LB is by shown in SEQ ID NO:7;
(2) sequence of described primer EFIP is by shown in SEQ ID NO:11, the sequence of described primer EBIP is by shown in SEQ ID NO:12, the sequence of described primers F 3 is by shown in SEQ ID NO:8, the sequence of described primer B3 is by shown in SEQ ID NO:9, the sequence of described primer LF is by shown in SEQ IDNO:13, and the sequence of described primer LB is by shown in SEQ ID NO:14.
9. method according to claim 8, is characterized in that, the fluorophor that in described combined sequence, primer EFIP sequence SEQ ID NO:4 marks is FAM, and the fluorophor that primer EFIP sequence SEQ IDNO:11 marks is HEX.
10. method according to claim 7, it is characterized in that, the goal gene fragment that described method can use one or more above step (2) described primers once to increase corresponding also detects, wherein, the fluorophor that the primer matched from each goal gene fragment marks is different, and demonstrates the result of otherness in the detection of step (4).
11. methods according to claim 10, is characterized in that, described primer sequence is one sequence combination:
(1) sequence of described primer EFIP is by shown in SEQ ID NO:4, the sequence of described primer EBIP is by shown in SEQ ID NO:5, the sequence of described primers F 3 is by shown in SEQ ID NO:1, the sequence of described primer B3 is by shown in SEQ ID NO:2, the sequence of described primer LF is by shown in SEQ IDNO:6, and the sequence of described primer LB is by shown in SEQ ID NO:7;
(2) sequence of described primer EFIP is by shown in SEQ ID NO:11, the sequence of described primer EBIP is by shown in SEQ ID NO:12, the sequence of described primers F 3 is by shown in SEQ ID NO:8, the sequence of described primer B3 is by shown in SEQ ID NO:9, the sequence of described primer LF is by shown in SEQ IDNO:13, and the sequence of described primer LB is by shown in SEQ ID NO:14;
The fluorophor that wherein described in two kinds of combined sequence, primer EFIP is labeled is respectively FAM or HEX.
12. 1 kinds of test kits detected for amplifying target genes fragment and to amplification, described test kit comprises:
(1) from 3 ' end of described goal gene fragment, set the first arbitrary sequence F1c, second arbitrary sequence F2c and the 3rd arbitrary sequence F3c, from 5 ' end of described goal gene fragment, set the 4th arbitrary sequence B1, during the 5th arbitrary sequence B2 and the 6th arbitrary sequence B3, primer EFIP is provided, described primer EFIP contains the sequence F2 with sequence F2c complementation, be connected with the sequence identical with sequence F1c at the 5 ' end of described sequence F2, be connected with one section of restriction enzyme identification fragment at described sequence F1c 5 ' end; There is provided primer EBIP, described primer EBIP contains sequence B 2, is connected with the sequence B 1c with sequence B 1 complementation at 5 ' end of described sequence B 2, is connected with one section of restriction enzyme identification fragment at described sequence B 1c 5 ' end; Wherein be marked with a fluorophor at the 5 ' end of primer EFIP and/or primer EBIP, have a quenching of fluorescence group at described other position mark be labeled in the primer of fluorophor except described restriction enzyme enzyme fragment, described quenching of fluorescence group can fluorophor described in quencher;
(2) chain shift-type polysaccharase, melting temperature(Tm) conditioning agent, primer, described restriction enzyme, damping fluid.
13. test kits according to claim 12, is characterized in that, described test kit also comprise containing with the primers F 3 of sequence F3c complementation, with containing the primer B3 identical with sequence B 3.
14. test kits according to claim 13, is characterized in that, described test kit also comprises pair of primers LF and LB containing complementary between F1c-F2c and between B1c-B2c.
15. test kits according to claim 14, described chain shift-type polysaccharase is Bst polysaccharase, described melting temperature(Tm) conditioning agent is trimethyl-glycine.
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