CN104962607B - A kind of single or multipurpose genetic fragment constant-temperature amplification detection method - Google Patents
A kind of single or multipurpose genetic fragment constant-temperature amplification detection method Download PDFInfo
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Abstract
The invention discloses a kind of single or multipurpose genetic fragment constant-temperature amplification detection method, the method passes through the end of FIB and/or BIP primers 5 ' the increase restriction enzyme site sequence in being expanded in LAMP, and mark fluorescent group on it, and mark quencher in primer other parts, the amplification of corresponding target gene can be shown according to fluoroscopic examination after amplification, so as to once expand the detection for completing single or a variety of target gene fragments, relative to traditional LAMP, this method can carry out single or Multiple detection, it is and efficient with amplification, rapid reaction, detection is special, the advantages of easy to operate.
Description
Technical field
The present invention relates to a kind of amplifying target genes fragment and the method that is detected to amplification, belong to molecular biosciences
Field.
Background technology
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) be by
A kind of new nucleic acid specificity amplification technique of the foundation such as Notomi, have high specificity, it is sensitive it is high, easy to operate, product is easy
The advantages that detection.The technology has been widely used for molecular diagnosis field.LAMP designs 4 for 6 specific regions of target sequence
Core primers, new chain synthesis are catalyzed using the Bst archaeal dna polymerases with strand-displacement activity, so that target sequence under constant temperature
Row efficient amplification.2 are inner primer, i.e. FIP (ForWard inner primer, FIP) and BIP among 4 core primers
(Backward inner primer, BIP).FIP includes Flc and F2 (complementary series in F2c regions), i.e. 5 '-Flc-F2；BIP
Include B1c (complementary series in B1 regions) and B2, i.e. 5 '-Blc-B2.Remaining two core primers is that outer primer is F3 and B3.Separately
Outer two ring primers (Loop primes, LF and LB), which are added in reaction system, accelerates LAMP reactions.
LAMP reactions include two processes, i.e. dumbbell shaped templated synthesis stage and cyclic amplification stage.The temperature of LAMP reactions
Spend for 60~65 DEG C, which is double-stranded DNA renaturation and the medium temperature of extension, and double-stranded DNA is within this temperature range in half
In the dynamic balance state of dissociation and quasi integration, any one primer carries out base pairing extension to the complementary portions of double-stranded DNA
When, another chain will dissociate, and become single-stranded.Under the action of strand displacement Bst archaeal dna polymerases, with 2 section of FIP primers Fs
3 ' ends are starting point, are matched with corresponding DNA complementary series, start strand displacement DNA synthesis.F3 primers and F2c front ends F3c sequences
Complementation, using 3 ' ends as starting point, by the effect of strand-displacement activity archaeal dna polymerase, displaces the DNA that FIP primers synthesize first
Chain, itself simultaneously synthesizing DNA.The DNA chain that final F3 primers are synthesized into forms double-strand with template DNA.It is but first by FIP primers
The DNA chain of synthesis is single-stranded by the progress strand displacement generation one of F3 primers, this is single-stranded mutual by the F1c sections and F1 sections of 5 ' ends
Mend, self base pairing occurs, form stem loop structure.Meanwhile BIP primers are combined with the single-stranded hybridization, with the 3 ' of BIP primers
Hold as starting point, synthesize complementary strand, loop-stem structure is opened in the process.Then, B3 primers and the complementary region on the outside of BIP primers
B3c pairings in domain combine, and are held with 3 ' as starting point, under the action of polymerase, synthesize new complementary strand.Pass through above-mentioned 2 processes, shape
Into double-stranded DNA.The single stranded DNA being replaced occurs self base pairing, forms ring-type knot according to complementary region existing for both ends
Structure, then whole DNA for being replaced out is in both ends presentation dumbbell structure, of the structure as LAMP reaction amplification cycles
Beginning structure.LAMP reacts the amplification cycles stage：First in dumbbell structure, using the Fl sections of 3 ' ends as starting point, with itself
For template, DNA synthesis extensions are carried out.At the same time, FIP primers Fs 2 and single-stranded F2c on ring hybridizes, and it is anti-to start new round strand displacement
Should, dissociate the double-strandednucleic acid synthesized by F1 sections.Equally, loop-stem structure can be also formed on the single-chain nucleic acid dissociateed.In stem
There are single stranded form B2c on ring structure, the B2 on BIP primers is hybrid with it, and starts new round amplification, by identical process,
Loop-stem structure is formed again.By this process, as a result complementary series forms structure not of uniform size in cycles on same chain.
LAMP reactions can specifically, it is efficient, quickly expand target DNA, reach 10 making the amount of product within an hour9A copy.
After LAMP amplifications, the detection of its product can be observed by agarose electrophoresis poststaining.Relatively simple method
It is directly to add SYBR Green I in the product to dye, it is positive reaction that green, which is presented, and orange red is negative reaction.Can also
The turbidity precipitated by expanding accessory substance magnesium pyrophosphate is judged that liquid is muddy, centrifuge or have white precipitate to be positive anti-
Should, no this phenomenon is then negative reaction.Present more simple method is to add visible dyes in the reactive mixture, positive
The color of reaction tube is changed into green from light gray, and negative reaction pipe then keeps original light gray.However, these methods all can only
Whether detection LAMP reactions carry out, it is impossible to which identification causes LAMP in testing goal sequence for the specific amplification of specific target sequence
During row, the judgement of its result lacks specificity.Therefore, traditional LAMP detections are difficult to realize to being examined while multipurpose fragment
Survey, this strongly limits the extensive use of LAMP.
In view of the above problems, some researchs are directed to developing multiple LAMP detection techniques.Realize that multiple LAMP detections are most normal
The method seen is that restriction enzyme digestion sites are found in target sequence, and LAMP products are used digestion with restriction enzyme,
It is corresponding according to different electrophoretic band sizes and corresponding target sequence by the postdigestive LAMP products of electrophoresis.However, this method
Two steps are needed to complete, during the LAMP products of restriction fragment different sizes, it is incomplete to take longer and digestion, leads
A target sequence is caused usually to correspond to several electrophoretic bands so that the result of multiple LAMP is difficult to be judged.Another kind is realized multiple
The new technology of LAMP detections is by LAMP amplified reactions and pyrosequencing combination.However, this method is situated between with restriction enzyme
The multiple LAMP detection techniques led are the same, are required for two steps to complete, and are LAMP amplifications first, are then corresponded to by pyrosequencing
To corresponding target sequence.This method is cumbersome, it is necessary to which specific kit purifies LAMP products, and sequencing procedure needs
Special personnel, and the sequenator and sequencing reagent that common lab can not be born.These inferior positions limit the popularization of this method
Use.In addition, existing multiple LAMP detection techniques can not all realize quick detection, complete multiple LAMP and detect time-consuming be more than
2.5 it is small when.Since the sensitivity of LAMP reactions is high, to later LAMP experiments, there are pole for the operation of uncapping of implementation LAMP products
Big pollution.
To sum up, no matter all there is be difficult to gram LAMP of the prior art in single amplification or the detection of multiplex amplification
The technical problem of clothes.
The content of the invention
The purpose of the present invention is improving the principle of LAMP reactions, based on LAMP reacts, with reference to restriction enzyme enzyme
Cut with fluoroscopic examination principle, establish the fast one-step method of a kind of easy to operate, high specificity, high sensitivity, detection speed it is single or
Multiple LAMP detections, to realize widely using for LAMP technology.
Based on above-mentioned purpose, present invention firstly provides a kind of amplifying target genes fragment and amplification is detected
Method, the described method comprises the following steps：
(1) risen from the 3 ' of target gene fragment ends, the first arbitrary sequence F1c, the second arbitrary sequence F2c of setting and the
Three arbitrary sequence F3c, rise from the 5 ' of target gene fragment ends, set the 4th arbitrary sequence B1, the 5th arbitrary sequence B2 with
6th arbitrary sequence B3；
(2) primer EFIP, the primer EFIP are provided and contains the sequence F2 with sequence F2c complementations, the sequence F2's
5 ' ends are connected with the sequence identical with sequence F1c, and one section of restriction enzyme identification piece is connected with the sequence F1c 5 ' ends
Section；There is provided primer EBIP, the primer EBIP contain the sequence identical with sequence B 2, the sequence B 25 ' end be connected with
The complementary sequence B 1c of sequence B 1, one section of restriction enzyme identification fragment is connected with the sequence B 1c 5 ' ends；Wherein drawing
5 ' the ends of thing EFIP and/or primer EBIP are marked with a fluorophor, except described in the primer of the labeled fluorophor
Other positions beyond restriction enzyme enzyme fragment are marked with a fluorescent quenching group, and the fluorescent quenching group can be quenched institute
State fluorophor；
(3) exist in chain shift-type polymerase, melting temperature conditioning agent, primer, the restriction enzyme and buffer solution
Under, application target genetic fragment is as template amplification DNA；
(4) amplification of detecting step (3).
Preferably, visible color change detection, electrophoresis detection or real-time fluorescence detection are detected as described in step (4).
Step (3) the chain shift-type polymerase can be that Bst archaeal dna polymerases, the melting temperature conditioning agent can be
Glycine betaine.
In a preferable technical solution, primer is further included containing complementary with sequence F3c provided in the step (2)
Primers F 3, and contain the primer B3 identical with sequence B 3.
Preferably, provided in the step (2) primer further include containing between F1c-F2c between B1c-B2c sequence
Arrange complementary pair of primers LF and LB.
Amplification can carry out in 60~66 DEG C described in step (2), in a preferable technical solution, step (2)
Described in amplification be to be carried out in 63~65 DEG C.
In a preferable technical solution, the primer sequence is combined selected from one sequence：
(1) sequence of the primer EFIP is by SEQ ID NO:Shown in 4, the sequence of the primer EBIP is by SEQ ID NO:
Shown in 5, the sequence of the primers F 3 is by SEQ ID NO:Shown in 1, the sequence of the primer B3 is by SEQ ID NO:Shown in 2, institute
The sequence of primer LF is stated by SEQ ID NO:Shown in 6, the sequence of the primer LB is by SEQ ID NO:Shown in 7；Or
(2) sequence of the primer EFIP is by SEQ ID NO:Shown in 11, the sequence of the primer EBIP is by SEQ ID
NO:Shown in 12, the sequence of the primers F 3 is by SEQ ID NO:Shown in 8, the sequence of the primer B3 is by SEQ ID NO:9 institutes
Show, the sequence of the primer LF is by SEQ ID NO:Shown in 13, the sequence of the primer LB is by SEQ ID NO:Shown in 14.
In above-mentioned combined sequence, detection of (1) group combination for Listeria monocytogenes bacterium, (2)
Detection of the group combination for Listeria ivanovii bacterium.
Preferably, primer EFIP sequence SEQ ID NO in the combined sequence:The fluorophors of 4 marks are FAM, primer
EFIP sequence SEQ ID NO:The fluorophor of 11 marks is HEX.
In presently preferred technical solution, the step of the method for the present invention can use one or more above
(2) primer once expands corresponding target gene fragment and is detected, wherein, with each target gene fragment phase
The fluorophor marked on matched primer is different, and the result of otherness is shown in the detection of step (4).
Preferably, the primer sequence combines for one sequence：
(1) sequence of the primer EFIP is by SEQ ID NO:Shown in 4, the sequence of the primer EBIP is by SEQ ID NO:
Shown in 5, the sequence of the primers F 3 is by SEQ ID NO:Shown in 1, the sequence of the primer B3 is by SEQ ID NO:Shown in 2, institute
The sequence of primer LF is stated by SEQ ID NO:Shown in 6, the sequence of the primer LB is by SEQ ID NO:Shown in 7；And
(2) sequence of the primer EFIP is by SEQ ID NO:Shown in 11, the sequence of the primer EBIP is by SEQ ID
NO:Shown in 12, the sequence of the primers F 3 is by SEQ ID NO:Shown in 8, the sequence of the primer B3 is by SEQ ID NO:9 institutes
Show, the sequence of the primer LF is by SEQ ID NO:Shown in 13, the sequence of the primer LB is by SEQ ID NO:Shown in 14.
In above-mentioned combined sequence, detection of (1) group combination for Listeria monocytogenes bacterium, (2)
Detection of the group combination for Listeria ivanovii bacterium, the ID of primer EFIP sequence SEQ described in two of which combined sequence
NO:The fluorophor of 4 marks is FAM, primer EFIP sequence SEQ ID NO:The fluorophor of 11 marks is HEX.
On the other hand, for amplifying target genes fragment and amplification is detected present invention also offers a kind of
Kit, the kit include：
(1) risen from the 3 ' of target gene fragment ends, the first arbitrary sequence F1c, the second arbitrary sequence F2c of setting and the
Three arbitrary sequence F3c, rise from the 5 ' of target gene fragment ends, set the 4th arbitrary sequence B1, the 5th arbitrary sequence B2 with
During the 6th arbitrary sequence B3, there is provided primer EFIP, the primer EFIP contains the sequence F2 with sequence F2c complementations, in the sequence
5 ' the ends of row F2 are connected with the sequence identical with sequence F1c, and one section of restriction enzyme is connected with the sequence F1c 5 ' ends
Identify fragment；Primer EBIP, the primer EBIP are provided and contain sequence B 2, is connected with and sequence B 1 at 5 ' ends of the sequence B 2
Complementary sequence B 1c, one section of restriction enzyme identification fragment is connected with the sequence B 1c 5 ' ends；Wherein in primer EFIP
And/or 5 ' the ends of primer EBIP are marked with a fluorophor, except described restricted in the primer of the labeled fluorophor
Other positions beyond internally-cut enzyme segment are marked with a fluorescent quenching group, and the fluorescent quenching group can be quenched the fluorescence
Group；
(2) chain shift-type polymerase, melting temperature conditioning agent, primer, the restriction enzyme, buffer solution.
Preferably, the kit is further included containing the primers F 3 with sequence F3c complementations, and containing identical with sequence B 3
Primer B3.
Preferably, the kit is further included containing a pair of sequence complementation is drawn between B1c-B2c between F1c-F2c
Thing LF and LB.
Preferably, the chain shift-type polymerase be Bst polymerases, the melting temperature conditioning agent be glycine betaine.
The present invention is improved the core primers FIP and/or BIP of LAMP on the basis of original LAMP primer,
5 ' one section of cleavage sequence (being named as Es) of end addition of FIP and/or BIP, which can be specific by restriction enzyme
Identification, improved primer are known as EFIP and EBIP.It should be noted that for the uniformity of statement, for anti-in the present invention
The above-mentioned core primers of system are answered, are whether added cleavage sequence, by Uniform Name are without exception in the claims EFIP
And EBIP.In specific embodiments of the present invention, for FIP and BIP how in the mode for specifically adding cleavage sequence, answer
It is subject to the specifically disclosed content of the embodiment, rather than is dependent only on the name title of the primer.Synthesis is for difference
During EFIP the and EBIP primers of target sequence, different fluorophors is marked at the 5 ' ends of EFIP and/or EBIP, is marked among primer
Remember quencher, which can be quenched 5 ' end fluorophors.
In the reaction system of the present invention, original FIP and BIP are substituted for EFIP and/or EBIP, the reaction is in perseverance
Carried out under the conditions of temperature, its reaction process is similar to LAMP reactions, with the progress of reaction, synthesized in reaction mixture using Es as
The complementary strand CEs of template, the double-strand can be by the specific identification of restriction enzyme, the cutting in reaction system, the processes
Fluorophor and quencher are separated, the fluorescence signal of release can be detected by fluorescence detector, different fluorescence signals
Represent different target sequences.Meanwhile after the Es sequences of EFIP/ or EBIP are sheared, which becomes the core of LAMP reactions
The heart primers F IP and BIP so that LAMP reactions continue (see Fig. 1).
The advantages of method (being named as MERT-LAMP technologies) provided by the invention, is embodied in：
1. compared with LAMP
For more common LAMP technology, when the MERT-LAMP technologies that the present invention establishes carry out unique sequence detection, its result
Interpretation can also use real-time fluorescence testing principle to carry out except that can carry out interpretation by conventional LAMP results read method
Interpretation, faster, operation is simpler for its detection rates.
The advantage of the present invention also resides in can carry out multisequencing detection in same reaction system, promote LAMP technology
Using.When carrying out multisequencing detection with MERT-LAMP, shorter detection time is consumed than LAMP, and detection sensitivity with
LAMP reactions are identical.
2. compared with existing multiple LAMP technology
For more existing multiple LAMP technology, the present invention can in same reaction tube a step, quickly finish more sequences
Row detection, it is not necessary to which electrophoresis or sequencing LAMP products, avoid opening LAMP reaction tubes, reduce the pollution to subsequent operation.
The result of MERT-LAMP technologies is read simply, and only the interpretation of fluorescence signal can need to be detected in real time.MERT-LAMP skills
Positive findings is presented in art, and most short take needs only to 12 minutes, most long 30 minutes time-consuming, faster than existing multiple LAMP technology
2.5 to 3 it is small when.In addition, the kit that MERT-LAMP technologies use is identical with common LAMP, it is not necessary to high sequencing reagent.
3. with qPCR Technical comparings
For qPCR technologies, the present invention has stronger Multiple detection ability, and EFIP and EBIP can be marked at the same time
Different luminophores.Based on the MERT-LAMP technologies of LAMP reactions, qPCR technologies are substantially better than in detection sensitivity, are examined
It is sensitive in qPCR technologies to survey 10 times of lower limit.In detection rates, MERT-LAMP technologies are equally better than qPCR technologies, MERT-LAMP
Positive findings is presented in technology, and most short take needs only to 12 minutes, is shortened about 20 minutes than qPCR, and each self-test is detected when at the same time
When surveying the DNA of threshold level, MERT-LAMP technologies need 30 minutes, and qPCR technologies then need 53 minutes.Therefore, it is of the invention
No matter in Multiple detection ability, detection rates, or qPCR technologies are better than in terms of test limit.
Brief description of the drawings
Fig. 1 .MERT-LAMP method testing principle schematic diagrames；
Fig. 2 .MERT-LAMP design of primers schematic diagrames；
Fig. 3 .MERT-LAMP amplification detects schematic diagrams；
The real-time Turbidity measurement schematic diagram of Fig. 4 .MERT-LAMP amplifications；
Fig. 5 different temperatures MERT-LAMP expands real-time fluorescence detects schematic diagram；
Fig. 6 substances MERT-LAMP expands real-time fluorescence detects schematic diagram；
The multiple MERT-LAMP amplifications real-time fluorescence detects schematic diagrams of Fig. 7；
The multiple MERT-LAMP methods Evaluation on specificity figures of Fig. 8.
Embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to protection scope of the present invention.
In order to verify the feasibility of method provided by the invention (MERT-LAMP technologies), two pathogenic Liszt's strains,
Listeria monocytogenes and listeria ivanovii are selected as model bacterium.It is special according to Listeria monocytogenes and listeria ivanovii kind
Specific gene lmo0733 (GenBank Gene ID:985919, Listeria monocytogenes, LM) and smcL
(GenBank Gene ID:11161600, Listeria ivanovii, Liv) two sets of MERT-LAMP primers of design,
Lmo0733-MERT-LAMP primers are used to detect Listeria monocytogenes, and smcL-MERT-LAMP primers are used to detect Yi Shi Li Si
Special bacterium.Lmo0733 is present in single increasing all serotypes of Liszt, and smcL is present in all hypotypes of listeria ivanovii,
Target gene it is specific good, can be by Listeria monocytogenes, Yi Shi Liszts and other closely similar strain (such as grignard Lee
This special bacterium, Weir Listeria, Ying Nuoke Listerias) and Pseudomonas (such as bacillus) distinguish.Set using LAMP primer
Count software PrimerExplorer V4 (Eiken Chemical) (http://primerexplorer.jp/e/) and primer set
Count software Primer Premier 5.0 and design MERT-LAMP primers, and by the specific primer of acquisition in ncbi database
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) in carry out sequence alignment analysis, to exclude primer and other
Species sequence non-specific matching that may be present, two sets of special MERT-LAMP amplimers after finally being optimized.Draw
The position and direction of thing design are shown in that (2-A is the MERT-LAMP primers for Listeria monocytogenes amplification to Fig. 2, and 2-B is for her
The MERT-LAMP primers of family name's Listeria amplification), sequence is shown in Table 1.
1 primer used in this application of table
(1) LM means L.monocytogenes：Listeria monocytogenes
(2) primer is not added with cleavage sequence, and it is to accelerate the reaction of MERT-LAMP to add the primer
(3) 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 acids, and BHQ-1 (Blackhole quencher1) refers to fluorescence quencher 1
(4) primer is not added with cleavage sequence, and it is to accelerate the reaction of MERT-LAMP to add the primer
(5) LI means L.ivanovii：Listeria ivanovii
(6) 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- methylfluoresceins
(7) F, sense primer
(8) R, anti-sense 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 arrive SEQ ID NO:26 be for two the sets of Listeria monocytogenes and listeria ivanovii commonly
LAMP primer, for comparing the sensitiveness of LAMP and MERT-LAMP, SEQ ID NO:27 arrive SEQ ID NO:32 are increased for single
Two sets of Real-time primers of Listeria and listeria ivanovii, for comparing the sensitiveness of qPCR and MERT-LAMP.
Used in the present invention and the reagent that is related to：
Loopamp Kit (Eiken Chemical Co.Ltd., Tokyo, Japan) are purchased from Japanese Rong Yan companies.DNA is carried
Take kit (QIAamp DNA minikits；Qiagen, Hilden, Germany) it is purchased from German Qiagen companies.QPCR is anti-
System mixture Premix (Takara Bio, Inc., Otsu, Japan, dNTP and buffer solution) is answered to be purchased from Beijing Takara biologies
Science and Technology Ltd..DL50DNA Marker and DL50DNA Marker are purchased from precious bioengineering (Dalian) Co., Ltd.Remaining
Reagent is commercially available parting net product.
The key instrument for using and being related in present invention experiment：The real-time transmissometer LA-320C (Eiken of Loopamp
Chemical Co., Ltd, Japan) it is purchased from Japanese Rong Yan companies.Real-time fluorescence detector is Rotor-Gene Q Real-
Time System (Qiagen), German Qiagen products；Electrophoresis equipment produces for Beijing Jun Yi east electrophoresis equipment Co., Ltd
Product；Gel imaging system is Bio-Rad Gel Dox XR, U.S.'s Bio-Rad products.
The extraction of the genomic DNA of Listeria monocytogenes, listeria ivanovii and other species bacterium：
Genome extracts：The extraction of bacterial genomes uses DNA extraction kit (the QIAamp DNA of Qiagen companies
minikits；Qiagen, Hilden, Germany), operated to specifications.Gene is measured using ultraviolet specrophotometer
The concentration and purity of group DNA, Listeria monocytogenes and listeria ivanovii genomic DNA with GE buffer serial-dilutions (from
25ng, 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 125fg to 62.5fg).Various genomic DNAs dispense on a small quantity, and -20
DEG C save backup.
The single amplifications of embodiment 1.MERT-LAMP：
(1) single amplification (i.e. standard reaction) systems of MERT-LAMP are as follows：The concentration of primer EFIP and FIP is respectively
The concentration of 20pmol, primer BIP are 40pmol, and the concentration of primers F 3 and B3 are 5pmol, and the concentration of primer LF and BF are
The MgSO of the Betain of 20pmol, 10mM, 6mM4, 10 × Bst DNA polymerase buffer liquid of the dNTP of 1mM, 2.5 μ l, 8U's
Strand displacement archaeal dna polymerase, the DNA restriction enzymes of 15U, the template of 1 μ l, adds deionized water to 25 μ l.Whole reaction exists
Carry out in fluorescence detector (Rotor-Gene Q Real Time System, Qiagen), isothermal duplication 63~65 DEG C of 1h, 80
DEG C 5min terminates reaction.
(2) feasibility is verified：
Under the conditions of standards system, MERT-LAMP primers and right are added according to designed by lmo0733 and smcL specific genes
The DNA profiling answered, lmo0733-MERT-LAMP reactions are used to expand Listeria monocytogenes (Fig. 3-A1,2,3-B1,2), smcl-
MERT-LAMP reactions are used to expand listeria ivanovii (Fig. 3-A3,4,3-B3,4), pass through visible color method of changing and electrophoresis is examined
Survey method confirms MERT-LAMP technical feasibilities.
Visible color method of changing：MERT-LAMP produces substantial amounts of pyrophosphate ion while synthetic DNA, the ion
The manganese ion combined with calcein can be captured, calcein is recovered free state and is fluoresced.The light-emitting admixture energy
It is enough to be combined with magnesium ion that is being produced in reaction, strengthened fluorescence.Fluorescence visual detection color change interpretation knot can be passed through
Fruit, positive reaction pipe are changed into green from light gray, and negative reaction keeps light grey constant, sees Fig. 3 A.Fig. 3-A1 are lmo0733-
The positive findings of MERT-LAMP primer amplification Listeria monocytogenes, Fig. 3-A2 are the negative knot of lmo0733-MERT-LAMP reactions
Fruit；Fig. 3-A3 are the positive findings of smcL-MERT-LAMP primer amplification listeria ivanoviis, and Fig. 3-A4 are smcL-MERT-
The negative findings of LAMP reactions., can be to target by visual detection the results show that the MERT-LAMP designed by the present invention is feasible
Sequence carries out augmentation detection.In addition, visual detection result also demonstrates two sets of the present invention according to designed by MERT-LAMP principles
Detected for the MERT-LAMP primers of Listeria monocytogenes and listeria ivanovii available for purpose bacterium.
Electrophoresis assays：Since MERT-LAMP reacts similar to LAMP reactions, its product is also a series of inverted repeats
The loop-stem structure and the DNA fragmentation mixture of polycyclic cauliflower spline structure that target sequence is formed, positive amplification is on gel after electrophoresis
Show the staged collection of illustrative plates of different size zone composition, negative findings occurs without amplified band, sees Fig. 3 B.Fig. 3-B1 are
The positive findings of lmo0733-MERT-LAMP primer amplification Listeria monocytogenes, Fig. 3-B2 react for lmo0733-MERT-LAMP
Negative findings；Fig. 3-B3 are the positive findings of smcL-MERT-LAMP primer amplification listeria ivanoviis, and Fig. 3-B4 are smcL-
The negative findings of MERT-LAMP reactions.Further proved by electrophoresis detection result, the MERT-LAMP skills designed by the present invention
Art is feasible, can carry out augmentation detection to target sequence.In addition, electrophoresis detection result also demonstrates the present invention according to MERT-LAMP
Two sets designed by principle are examined for the MERT-LAMP primers of Listeria monocytogenes and listeria ivanovii available for purpose bacterium
Survey.
Real-time fluorescence detection method：
Under the conditions of standards system, MERT-LAMP primers and right are added according to designed by lmo0733 and smcL specific genes
The DNA profiling answered, the reaction of lmo0733-MERT-LAMP real-time amplifications are used for the detection to purpose bacterium Listeria monocytogenes, smcL-
The reaction of MERT-LAMP real-time amplifications is used for the detection to purpose bacterium listeria ivanovii.Under the conditions of standards system, according to MERT-
LAMP reaction principles and designed primer, interpretation as a result rely on the different fluorescence signals produced after digestion, are examined by fluorescence
Device is surveyed to receive to obtain stable fluorescence curve (Fig. 4).Fig. 4 A are lmo0733-MERT-LAMP primers (FAM marks) real-time amplification
Testing result, Fig. 4-A1 are the positive findings of detection Listeria monocytogenes, and amplified signal is more than threshold value, and Fig. 4-A2 are negative control
As a result, any amplification curve is not produced.Fig. 4 B are smcL-MERT-LAMP primers (HEX marks) real-time amplification testing result, figure
4-B1 is the positive findings of detection listeria ivanovii, and amplified signal is more than threshold value, and Fig. 4-B2 are negative control as a result, not producing
Any amplification curve.Being proved by real-time fluorescence testing result, MERT-LAMP results can carry out interpretation by real-time fluorescence,
So as to provide foundation for the interpretation of Multiple detection result, secondly fluoroscopic examination result is further demonstrated designed by the present invention
MERT-LAMP technical feasibilities, can carry out augmentation detection to target sequence.In addition, real-time fluorescence testing result also demonstrates this hair
Bright two sets of MERT-LAMP primers for being directed to Listeria monocytogenes and listeria ivanovii according to designed by MERT-LAMP principles
Detected available for purpose bacterium.
(3) optimal reaction temperature of MERT-LAMP technologies is measured
Under the conditions of standards system, the DNA moulds of listeria ivanovii MERT-LAMP primers and listeria ivanovii are added
Plate, its template concentrations are 2.5pg/ μ l.Reaction carries out (61~66 DEG C) under constant temperature, application of results real-time fluorescence detector
It is detected, obtains different dynamic curve diagrams at different temperature, see Fig. 5.Fig. 5 A to 5F are respectively MERT-LAMP amplifications
The amplification under the conditions of 61~66 DEG C is reacted, under 63~65 DEG C of amplification conditions, MERT-LAMP amplification curves are stablized, and go out
The time of existing positive findings is early, therefore 63~65 DEG C of optimal reaction temperatures for being proposed as MERT-LAMP technologies.The present invention's
Follow-up study selection carries out under the conditions of 64 DEG C.
(4) MERT-LAMP detects the sensitivity of single sample
Under the conditions of standards system, serial dilution Listeria monocytogenes and listeria ivanovii template DAN (2.5ng,
250pg, 25pg, 2.5pg, 250fg, 125fg and 62.5fg/ microlitre), 1 microlitre of each concentration template is added in reaction system, is passed through
Real-time fluorescence detects, and obtains stable fluoroscopic examination figure, sees Fig. 6.6A is lmo0733-MERT-LAMP primers (FAM marks)
Real-time amplification testing result, 6-A1 to 6-A5 amplified signals curve are more than threshold value, and interpretation is the positive of detection Listeria monocytogenes
As a result, corresponding Listeria monocytogenes template concentrations are (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 6-A6 is arrived
6-A8, amplified signal curve are less than threshold value, and to detect negative findings, corresponding Listeria monocytogenes template concentrations are 125fg,
62.5fg/ microlitres and negative control pipe, therefore the Monitoring lower-cut of MERT-LAMP technology for detection Listeria monocytogenes nucleic acid is
250fgDNA/ reaction tubes.6B is smcL-MERT-LAMP primers (HEX marks) real-time amplification testing result, and 6-B1 to 6-B5 expands
Increase signal curve and be more than threshold value, to detect the positive findings of listeria ivanovii, corresponding listeria ivanovii template is dense for interpretation
Spend and be less than threshold value for (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 6-A6 to 6-A8, amplified signal curve, for inspection
Negative findings is surveyed, corresponding listeria ivanovii template concentrations be 125fg, 62.5fg/ microlitres and feminine gender control pipe, therefore
The Monitoring lower-cut of MERT-LAMP technology for detection listeria ivanovii nucleic acid is 250fg DNA/ reaction tubes.
According to Fig. 6 presentations as a result, MERT-LAMP technologies are anti-for 250fg DNA/ for the Monitoring lower-cut of single target sequence
Ying Guan, and the most short detection time consumed is about 12 minutes, the DNA of detection minimum detection limit level also only consumes about 30
Minute.When genomic templates amount is reduced to below 250fg in reaction system, MERT-LAMP reactions occur without positive amplification.Root
According to interpretation as a result, the remolding sensitivity qPCR methods of MERT-LAMP amplified reactions are 10 times high, 2 are shown in Table.
Embodiment 2.MERT-LAMP multiplex amplifications
(1) MERT-LAMP multiple reactions system is as follows：The concentration of primer EFIP and FIP are respectively 20pmol, primer BIP's
Concentration is 40pmol, and the concentration of primers F 3 and B3 is 10pmol, and the concentration of primer LF and BF is 10pmol, the Betain of 10mM,
The MgSO of 6mM4, 10 × Bst DNA polymerase buffer liquid of the dNTP of 1mM, 2.5 μ l, the strand displacement archaeal dna polymerase of 8U, 15U's
DNA restriction enzymes, the template of 1 μ l, adds deionized water to 25 μ l.Entirely react in fluorescence detector (Rotor-Gene
Q Real Time System, Qiagen) in carry out, for isothermal duplication in 64 DEG C of 1h, 80 DEG C of 5min terminate reaction.
(2) Multiple detection ability and the sensitivity of MERT-LAMP is verified
In order to realize that the multiplex amplification of MERT-LAMP technologies detects, the present invention optimizes standards system more to adapt to
Re-detection, establishes Multiple detection system.Under Multiple detection system, two sets of primers and right are added at the same time in multiple reaction mixture
The template answered, is detected by fluorescence detection equipment, obtains stable multi-fluorescence detection figure, sees Fig. 7,7A and 7B real-time amplification song
Line is generated at the same time, and the amplification curve of 7A comes from FAM sense channels, represents lmo0733-MERT-LAMP primers (FAM marks)
Real-time amplification testing result, 7-A1 to 7-A5 amplified signals curve are more than threshold value, and interpretation is the positive of detection Listeria monocytogenes
As a result, corresponding Listeria monocytogenes template concentrations are (2.5ng, 250pg, 25pg, 2.5pg and 250fg/ microlitre), 7-A6 is arrived
7-A8, amplified signal curve are less than threshold value, and to detect negative findings, corresponding Listeria monocytogenes template concentrations are 125fg,
62.5fg/ microlitres and negative control pipe, therefore, when carrying out Multiple detection with MERT-LAMP technologies, to Listeria monocytogenes
The Monitoring lower-cut of nucleic acid is 250fg DNA/ reaction tubes.The amplification curve of 7B comes from HEX sense channels, represents smcL-MERT-
LAMP primer (HEX marks) real-time amplification testing result, 7-B1 to 7-B5 amplified signals curve are more than threshold value, and interpretation is to detect her
The positive findings of family name's Listeria, corresponding listeria ivanovii template concentrations for (2.5ng, 250pg, 25pg, 2.5pg and
250fg/ microlitres), 7-B6 to 7-B8, amplified signal curve is less than threshold value, to detect negative findings, corresponding listeria ivanovii
Template concentrations are 125fg, and 62.5fg/ microlitres and negative control pipe, therefore, Multiple detection are being carried out with MERT-LAMP technologies
When, the Monitoring lower-cut to listeria ivanovii nucleic acid is 250fg DNA/ reaction tubes.It is anti-from the result of Fig. 7, MERT-LAMP
System is answered to be carried out at the same time the detection of two target sequences, it was demonstrated that the Multiple detection abilities of MERT-LAMP technologies, can will
Detection of the MERT-LAMP Technique Popularizings to more aim sequences.When detecting multiple target sequences using MERT-LAMP, its detection time
The change of no matter is detected with detection sensitivity and simple sequence, shortest detection time is 12 minutes or so, the spirit of two sets of templates
Sensitivity is 250fg DNA/ reaction tubes, is shown in Table 2.
Detection of the different amplification technique of the application of table 2 to listeria ivanovii is compared
(3) Evaluation on specificity：
With the genomic DNA of common pathogenic bacteria and conditioned pathogen (Listeria monocytogenes, listeria ivanovii, Ying Nuo
Gram Listeria, Weir Listeria, Xi Er Listerias, Listera grayi, Bacillus cereus, the pathogenic large intestine bar of intestines
Bacterium, enterotoxigenic E.Coli, enteroinvasive E.Coli etc.) as template evaluation MERT-LAMP reaction systems specificity,
It is shown in Table 3.Two sets of MERT-LAMP primers (lmo0733-MERT-LAMP primers, smcL-MERT-LAMP primers) are added at the same time
In MERT-LAMP multiplex amplification systems, augmentation detection is then carried out, the genomic DNA template that amplified reaction pipe 1 to 12 adds is
The Listeria monocytogenes (1/2a, 3a, 1/2b, 3b, 7,1/2c, 3c, 4a, 4c, 4b, 4d and 4e) of 12 serotype, reaction tube 13,
14 genomic DNA template is listeria ivanovii reference culture and listeria ivanovii isolated strains, reaction tube 15 to 35 are
The genomic DNA template of other bacteriums, reaction tube 36 are negative control pipe, and the result is shown in Fig. 8 for specificity identification.8A and 8B expand in real time
Increase curve to be generated at the same time, the amplification curve of 8A comes from FAM sense channels, represents lmo0733-MERT-LAMP primers (FAM
Mark) real-time amplification testing result, 8-A1 to 8-A12 amplified signals curve is more than threshold value, and interpretation is to detect Listeria monocytogenes
Positive findings, corresponding genomic DNA template for 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, corresponding to detect negative findings
Genomic DNA template is listeria ivanovii, other bacteriums and negative control pipe；It is logical that the amplification curve of 8B comes from HEX detections
Road, represents smcL-MERT-LAMP primers (HEX marks) real-time amplification testing result, and 8-B13 to 8-B14 amplified signal curves are big
In threshold value, interpretation is the positive findings of detection listeria ivanovii, and corresponding genomic DNA template is listeria ivanovii standard
Bacterial strain and listeria ivanovii isolated strains；8-B1 to 8-B12,8-B15 to 8-B36 amplified signal curve are less than threshold value, interpretation
For negative result, the corresponding genomic DNA templates of 8-B1 to 8-B12 for 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 that other are thin
Bacterium and negative control pipe；Therefore, MERT-LAMP technologies can accurately differentiate Listeria monocytogenes and listeria ivanovii, explanation
The specificity of MERT-LAMP methods is good.
3 bacterial strain uses therefor of the present invention of table
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 systems
Wherein, the upstream primer sequence for Listeria monocytogenes detection is SEQ ID NO:27, downstream primer sequence is
SEQ ID NO:28, probe sequence is SEQ ID NO:29, the upstream primer sequence for listeria ivanovii detection is SEQ
ID NO:30, downstream primer sequence is SEQ ID NO:31, probe sequence is SEQ ID NO:32.
Amplification and measurement result on real-time fluorescence quantitative PCR instrument：
It is anti-that Real-time is carried out in fluorescence detector (Rotor-Gene Q Real Time System, Qiagen)
Should, after amplification, same Threshold Analysis data are taken after deducting background fluorescence signal, determine the Ct (cycle of the reaction
Threshold) value.
Common LAMP reaction systems：
The reaction system of LAMP：Cumulative volume is 25 μ l, comprising 40pmol inner primers FIP and BIP, 5pmol outer primer F3 and
B3,20pmol ring primer LF and BF, 20mM Tris-HCl (pH 8.8), 10mM KCl, 4mM MgSO4,10mM(NH4)2SO4,
0.1%Tween 20,0.8M glycine betaines, 1.4mM dNTPs, 1 μ l Bst archaeal dna polymerases (8U/mL), 1 μ l FD reagents (Lang Pu
Fluorescent visual reagent) and 1 μ l DNA profilings.The reaction condition of LAMP is all 64 DEG C of constant temperature, reaction time 60min.80℃
5min terminates reaction.
Wherein, the FIP for Listeria monocytogenes detection is SEQ ID NO:17, BIP be SEQ ID NO:18, F3 are
SEQ ID NO:15, B3 be SEQ ID NO:16, LF be SEQ ID NO:19, LB be SEQ ID NO:20.For Yi Shi Li Si
The FIP of special bacterium detection is SEQ ID NO:23, BIP be SEQ ID NO:24, F3 be SEQ ID NO:21, B3 be SEQ ID NO:
22, LF be SEQ ID NO:25, LB be SEQ ID NO:26.
Claims (15)
1. a kind of ring mediated isothermal amplification target gene fragment for non-diagnostic purpose and the side being detected to amplification
Method, the described method comprises the following steps：
(1) risen from the 3 ' of target gene fragment ends, set the first arbitrary sequence F1c, second arbitrary sequence F2c and the 3rd
Anticipate sequence F3c, from 5 ' ends of the target gene fragment, sets the 4th arbitrary sequence B1, the 5th arbitrary sequence B2 and the 6th
Arbitrary sequence B3；
(2) primer EFIP, the primer EFIP are provided and contains the sequence F2 with sequence F2c complementations, at the 5 ' ends of the sequence F2
The sequence identical with sequence F1c is connected with, one section of restriction enzyme identification fragment is connected with the sequence F1c 5 ' ends；Carry
For primer EBIP, the primer EBIP contains sequence B 2, and the sequence complementary with sequence B 1 is connected with 5 ' ends of the sequence B 2
B1c, one section of restriction enzyme identification fragment is connected with the sequence B 1c 5 ' ends；Wherein in primer EFIP and/or primer
5 ' the ends of EBIP are marked with a fluorophor, and the restriction enzyme enzyme fragment is removed in the primer of the labeled fluorophor
Other positions in addition are marked with a fluorescent quenching group, and the fluorescent quenching group can be quenched the fluorophor；
(3) in the presence of chain shift-type polymerase, melting temperature conditioning agent, primer, the restriction enzyme and buffer solution, make
Template amplification DNA is used as by the use of target gene fragment；
(4) amplification of detecting step (3).
2. according to the method described in claim 1, it is characterized in that, it is detected as the inspection of visible color method of changing described in step (4)
Survey, electrophoresis detection or real-time fluorescence detect.
3. according to the method described in claim 1, it is characterized in that, the chain shift-type polymerase be Bst archaeal dna polymerases, institute
It is glycine betaine to state melting temperature conditioning agent.
4. according to the method described in claim 1, it is characterized in that, provided in the step (2) primer further include containing with
The primers F 3 of sequence F3c complementations, and contain the primer B3 identical with sequence B 3.
5. according to the method described in claim 1, it is characterized in that, provided in the step (2) primer further include containing with
Between F1c-F2c between B1c-B2c sequence complementation pair of primers LF and LB.
6. the method according to any claim in Claims 1 to 5, it is characterised in that being expanded described in step (3) is
Carried out in 60~66 DEG C.
7. according to the method described in claim 6, it is characterized in that, amplification described in step (3) is carried out in 63~65 DEG C.
8. the method according to the description of claim 7 is characterized in that the primer sequence is combined selected from one sequence:
(1) sequence of the primer EFIP is by SEQ ID NO:Shown in 4, the sequence of the primer EBIP is by SEQ ID NO:5 institutes
Show, the sequence of the primers F 3 is by SEQ ID NO:Shown in 1, the sequence of the primer B3 is by SEQ ID NO:It is described to draw shown in 2
The sequence of thing LF is by SEQ ID NO:Shown in 6, the sequence of the primer LB is by SEQ ID NO:Shown in 7；
(2) sequence of the primer EFIP is by SEQ ID NO:Shown in 11, the sequence of the primer EBIP is by SEQ ID NO:12
Shown, the sequence of the primers F 3 is by SEQ ID NO:Shown in 8, the sequence of the primer B3 is by SEQ ID NO:It is described shown in 9
The sequence of primer LF is by SEQ ID NO:Shown in 13, the sequence of the primer LB is by SEQ ID NO:Shown in 14.
9. according to the method described in claim 8, it is characterized in that, primer EFIP sequences SEQ ID NO in the combined sequence:
The fluorophor of 4 marks is FAM, primer EFIP sequence SEQ ID NO:The fluorophor of 11 marks is HEX.
10. the method according to the description of claim 7 is characterized in that the method can use the step more than one or more
Suddenly (2) described primer once expands corresponding target gene fragment and is detected, wherein, with each target gene fragment
The fluorophor marked on the primer to match is different, and the result of otherness is shown in the detection of step (4).
11. according to the method described in claim 10, it is characterized in that, the primer sequence combines for one sequence：
(1) sequence of the primer EFIP is by SEQ ID NO:Shown in 4, the sequence of the primer EBIP is by SEQ ID NO:5 institutes
Show, the sequence of the primers F 3 is by SEQ ID NO:Shown in 1, the sequence of the primer B3 is by SEQ ID NO:It is described to draw shown in 2
The sequence of thing LF is by SEQ ID NO:Shown in 6, the sequence of the primer LB is by SEQ ID NO:Shown in 7；
(2) sequence of the primer EFIP is by SEQ ID NO:Shown in 11, the sequence of the primer EBIP is by SEQ ID NO:12
Shown, the sequence of the primers F 3 is by SEQ ID NO:Shown in 8, the sequence of the primer B3 is by SEQ ID NO:It is described shown in 9
The sequence of primer LF is by SEQ ID NO:Shown in 13, the sequence of the primer LB is by SEQ ID NO:Shown in 14；
The fluorophor that primer EFIP is labeled described in two of which combined sequence is respectively FAM or HEX.
12. a kind of kit for being used for ring mediated isothermal amplification target gene fragment and being detected to amplification, the examination
Agent box includes：
(1) risen from the 3 ' of target gene fragment ends, set the first arbitrary sequence F1c, second arbitrary sequence F2c and the 3rd
Anticipate sequence F3c, from 5 ' ends of the target gene fragment, sets the 4th arbitrary sequence B1, the 5th arbitrary sequence B2 and the 6th
During arbitrary sequence B3, there is provided primer EFIP, the primer EFIP contain the sequence F2 with sequence F2c complementations, in the sequence F2
5 ' ends be connected with the sequence identical with sequence F1c, be connected with the identification of one section of restriction enzyme at the sequence F1c 5 ' ends
Fragment；Primer EBIP, the primer EBIP are provided and contain sequence B 2, is connected with 5 ' ends of the sequence B 2 complementary with sequence B 1
Sequence B 1c, one section of restriction enzyme identification fragment is connected with the sequence B 1c 5 ' ends；Wherein primer EFIP and/
Or 5 ' the ends of primer EBIP are marked with a fluorophor, and the restriction enzyme is removed in the primer of the labeled fluorophor
Other positions beyond enzyme fragment are marked with a fluorescent quenching group, and the fluorescent quenching group can be quenched the fluorescent base
Group；
(2) chain shift-type polymerase, melting temperature conditioning agent, primer, the restriction enzyme, buffer solution.
13. kit according to claim 12, it is characterised in that the kit is further included containing mutual with sequence F3c
The primers F 3 of benefit, and contain the primer B3 identical with sequence B 3.
14. kit according to claim 13, it is characterised in that the kit further include containing with F1c-F2c it
Between between B1c-B2c sequence complementation pair of primers LF and LB.
15. kit according to claim 14, the chain shift-type polymerase is Bst polymerases, the melting temperature
Conditioning agent is glycine betaine.
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| CN105219772B (en) * | 2015-11-15 | 2018-04-20 | 中国疾病预防控制中心传染病预防控制所 | One group of nucleotide sequence and the application in detection of Salmonella and shigella dysenteriae detection |
| CN105349526B (en) * | 2015-11-22 | 2018-10-02 | 中国疾病预防控制中心传染病预防控制所 | A kind of method and application carrying out constant-temperature amplification nucleic acid using multiple inner primer |
| KR101875662B1 (en) | 2015-12-01 | 2018-08-02 | 에스디 바이오센서 주식회사 | An isothermal based-dual functional oligonucleotide including reporter dye, and quencher for isothermal nucleic acid amplification and measurement methods using same |
| CN105506121A (en) * | 2016-01-10 | 2016-04-20 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence used for vibrio parahaemolyticus and vibrio vulnificus detection |
| CN105755134B (en) * | 2016-04-11 | 2020-02-04 | 中国疾病预防控制中心传染病预防控制所 | Endonuclease-mediated real-time multiple cross-displacement nucleic acid amplification technology and application |
| US11168357B2 (en) | 2016-05-24 | 2021-11-09 | Atila Biosystems, Inc. | Omega amplification |
| CN106048015A (en) * | 2016-06-07 | 2016-10-26 | 中国疾病预防控制中心传染病预防控制所 | ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology |
| CN105950778A (en) * | 2016-07-20 | 2016-09-21 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence for detecting enterococcus faecalis and staphylococcus aureus with MERT-LAMP technology |
| CN106893787A (en) * | 2017-04-20 | 2017-06-27 | 广西壮族自治区兽医研究所 | Differentiate bifluorescence RT LAMP detection primers group, kit and its application of foot and mouth disease virus and vesicular stomatitis virus |
| CN111197094B (en) * | 2018-11-16 | 2021-02-23 | 深圳市疾病预防控制中心 | Compositions, kits and methods for genotyping vibrio parahaemolyticus |
| CN110184367B (en) * | 2019-06-11 | 2022-07-08 | 扬州大学 | Multiple PCR detection kit for Listeria monocytogenes and Listeria monocytogenes |
| CN114317828A (en) * | 2022-01-14 | 2022-04-12 | 广州朗坤生物科技有限公司 | LAMP primer combination, reaction system and method for detecting HPV types 52 and 58 |
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