CN109055500A - A kind of fluorescence ring mediated isothermal amplification method based on molecular beacon - Google Patents
A kind of fluorescence ring mediated isothermal amplification method based on molecular beacon Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence ring mediated isothermal amplification method based on molecular beacon.Molecular beacon is introduced into LAMP technology by the present invention, establish the fluorescence ring mediated isothermal amplification method based on molecular beacon, and it is named as MB-LAMP, to realize the direct detection to amplified production, it fundamentally solves the problems, such as LAMP method non-specific amplification, there is huge application value and commercial value.
Description
Technical field
The present invention relates to a kind of fluorescence ring mediated isothermal amplification method based on molecular beacon.
Background technique
Loop-mediated isothermal amplification technique is to detect skill by a kind of novel in vitro nucleic acid amplification that Notomi was established in 2001
Art.It removes the nucleic acid fragment of 6-8 sections of specificity of identification using 4-6 primer.Archaeal dna polymerase has the function of strand displacement, it
Under help, target gene can obtain efficient, special amplification in a short period of time, under conditions of constant temperature.At the same time,
A large amount of white magnesium pyrophosphate precipitating is precipitated in reaction, and using this feature, experimenter can pass through the muddy of observing response system
It is negative or positive that turbid degree, which carrys out judging result,.Since LAMP reaction carries out under constant temperature conditions, so a stabilization
Heat source (such as water-bath or metal bath) be just able to satisfy the requirement of experiment condition, at the same time, the instrument expense of experiment is also big
It reduces greatly.Due to the plurality of advantages of above-mentioned LAMP, it is in microorganism detection, such as bacterium, fungi, viral and gene
Disease detection, baby's early sex diagnosis etc. have obtained universal application.
PCR is two regions of using two primer specifics identifying purpose segment, and LAMP is to identify 6-8 independently
Genetic fragment, therefore theoretically, LAMP should have higher specificity than PCR, but true really not so.Cause
It is more for primer number, so LAMP is easier to encounter by non-specific amplification and bring false positive issue, this point by
Several workers are confirmed.And importantly, due to traditional detection method be all indirectly to reflection product detect,
So this false positive cannot be told in true result.Traditional detection method principle approximately as:
1, electrophoresis: strip analysis is carried out using reflection product of traditional gel electrophoresis to LAMP.But due to
It is irregular that LAMP, which amplifies the clip size come, so electrophoresis result is in diffuse state.No matter which serial primer or
Template participates in reaction, and the result of electrophoresis is all almost similar, so we cannot determine whether there is according to the shapes and sizes of band
The result of false positive generates.The mode of electrophoresis is easy to generate Aerosol Pollution simultaneously, this will will affect the experimental result of LAMP.
2, I decoration method of SYBR Green: SYBR Green I can be swashed after being integrated in DNA double chain structure by ultraviolet light
It sends out and shines.Using this characteristic, reacting in LAMP terminates that SYBR Green I is added dropwise into product with rear, we can be
Under ultraviolet light according to green fluorescence interpretation experimental result.But in addition to amplified production, SYBR Green I can be in conjunction with all
Double-stranded DNA and shine, to cause the case where result is not easy interpretation, high fluorescence background value.In addition to this, due to SYBR
Green I, which needs to open reaction tube after the completion of reaction, to be added, and reflection in this way cannot carry out in an enclosed system, from
And it can there is a situation where Aerosol Pollutions.
3, the precipitation method: magnesium pyrophosphate precipitating can be generated in LAMP reaction process, and the amount generated is bigger, reaction is completed
Afterwards by the effect of centrifugation, all precipitatings can be flocked together, observe by the naked eye can judging result, but due to coke
Magnesium phosphate has extraordinary dispersibility in solution system, can be dispersed back into reaction solution with moment after centrifugation aggregation,
Thus it is easy to produce false negative.
4, hydroxynaphthol blue dyeing (HNB): hydroxynaphthol blue dyeing is a kind of Metal ion indicator, it can be according to solution
Middle Mg2+Difference and show different colors, due to LAMP reaction in generate magnesium pyrophosphate precipitating, can largely consume Mg2+,
The color of HNB will change in this way, but HNB is unstable, it is not easy to and it saves, needs matching while using, use inconvenience, it is another
A problem is that the color reaction of HNB is unobvious, it is not easy to color change is discovered, although Many researchers are using we at present
Method, but everybody approval is not obtained.
5, nephelometry: nephelometry is that current research LAMP reaction obtains the most method of approval, it passes through real-time transmissometer
The variation of reaction system turbidity is detected, and draws curve to reflect reaction result, since whole process is monitored by instrument, eliminates people
For the interference of judgement, the time point that credible result, furthermore this method can be reacted with true reappearance LAMP, the ginseng such as reaction efficiency
Number has extraordinary directive significance for the experiment of best primer screening, optimum temperature experiment, is being that research and development LAMP kit must
Indispensable instrument.
6, calcein/Mn2+Dyeing: calcein itself is Metal ion indicator, can be according to metal ion in solution
Number and issue fluorescence, Mn2+It can be chelated with calcein, and then its fluorescence is quenched, can generated in LAMP reaction process
Magnesium pyrophosphate precipitating, if containing Mn in reaction solution2+If, then what is generated is manganese pyrophosphate precipitating, can thus release
Release the Mn with calcein chelating2+, the fluorescence of calcein also just is excited out.
7, complex probe: according to fluorescence energy transfer principle, Chen Suhong etc. designs and has synthesized a kind of new compound spy
Needle, the probe are made of a long fluorogenic hybridization probe and short quenching probes, and wherein fluorescence probe 5 ' terminates a fluorescein
Molecule, 3 ' termination one extend blocker molecule phosphoric acid, quenching probes 3 ' end connection one quencher molecule paramethyl red, quenching probes with
The end of fluorescence probe 5 ' is complementary, and when no template, which hybridizes to form combined probe, and unstressed configuration generates, and when there is template, fluorescence is visited
Needle hybridizes with template, and fluorescence cannot be quenched, and the fluorescence of generation is directly proportional to template quantity.Although combined probe introduces probe,
But it is not still the direct detection to product.
These Indirect Detecting Methods can not directly determine amplified production, if producing non-specific amplification,
It will be unable to identify, this is also the big problem that LAMP technology is met with.
Summary of the invention
The object of the present invention is to provide a kind of fluorescence ring mediated isothermal amplification method based on molecular beacon.
The present invention protects a kind of primer combination of probe for fluorescence ring mediated isothermal duplication first, by LAMP primer composition
It is formed with molecular beacon probe LBP；The LAMP primer composition by preceding primer F3, rear primer B3, preceding inner primer FIP, it is rear in draw
Object BIP and acceleration primer LB composition；
To accelerate primer LB people is to be divided into LB-a section and LB-b section；Molecular beacon probe LBP is from upstream to downstream
It successively include: LBP-b1 section, LBP-a section and LBP-b2 section；The nucleotide sequence of LBP-a section and the core of LB-a section
Nucleotide sequence is identical；LBP-b1 section and LBP-b2 section reverse complemental；The nucleotides sequence of LBP-b1 section or LBP-b2 section
It arranges identical as the nucleotide sequence of LB-b section；
The molecular beacon probe LBP, an end are connected with fluorophor, another end is connected with fluorescent quenching base
Group.
The LBP-b1 section is made of 6 nucleotide；The LBP-a section is made of 12-13 nucleotide.
The LBP-a section can be specifically made of 13 nucleotide.
The fluorophor can specifically be located at 5 ' ends of molecular beacon probe.
It is described to have fluorescent quenching group specifically be positioned at 3 ' ends of molecular beacon probe.
The fluorophor concretely fluorophor FAM.
The fluorescent quenching group concretely fluorescent quenching group DABCYL.
The primer combination of probe concretely primer combination of probe first, in the primer combination of probe first,
The preceding primer F3 is following (a1) or (a2)；
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of identical function；
Primer B3 is following (a3) or (a4) after described；
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of identical function；
The preceding inner primer FIP is following (a5) or (a6):
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) sequence 3 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3
The DNA molecular of identical function；
Inner primer BIP is following (a7) or (a8) after described:
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) sequence 4 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4
The DNA molecular of identical function；
The acceleration primer LF is following (a9) or (a10):
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) sequence 5 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5
The DNA molecular of identical function；
The acceleration primer LB is following (a11) or (a12):
(a11) single strand dna shown in the sequence 6 of sequence table；
(a12) sequence 6 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6
The DNA molecular of identical function；
The molecular beacon probe LBP is following (b1) or (b2):
(b1) single strand dna shown in the sequence 7 of sequence table；
(b2) sequence 7 is had by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 7
The DNA molecular of identical function.
The present invention also protects application of the primer combination of probe of any description above in reagent preparation box；The kit
Function be (c1) or (c2):
(c1) whether detection sample to be tested is object；
(c2) detect in sample to be tested whether contain object.
The present invention also protects the kit containing any description above primer combination of probe；The function of the kit is
(c1) or (c2):
(c1) whether detection sample to be tested is object；
(c2) detect in sample to be tested whether contain object.
The present invention also protects the preparation method of the kit, includes the steps that individually packing each primed probe.
The present invention also protect it is a kind of detection sample to be tested whether be object method, include the following steps: to test sample
This genomic DNA or cDNA is template, carries out fluorescence ring mediated isothermal expansion using the primer combination of probe of any description above
Increase, monitor fluorescence signal, if showing that positive amplification curve, sample to be tested are or candidate is object, if not showing the positive
Amplification curve, sample to be tested are or candidate is non-targeted object.
The present invention also protect in a kind of detection sample to be tested whether the method containing object, include the following steps: with to
The cDNA that the total serum IgE reverse transcription of the total DNA or sample to be tested of test sample sheet obtains is template, is visited using the primer of any description above
Needle combination carries out fluorescence ring mediated isothermal duplication, monitors fluorescence signal, if display positive amplification curve, sample to be tested contain or
It is doubtful containing object, if not showing that positive amplification curve, sample to be tested do not contain or doubtful without containing object.
In the reaction system of the fluorescence ring mediated isothermal duplication of any description above, the concentration of the molecular beacon probe LBP
It can be 2-10pmol, concretely 2pmol, 4pmol, 6pmol, 8pmol or 10pmol, most preferably 8pmol.
The reaction temperature of the fluorescence ring mediated isothermal duplication of any description above can be 60-66 DEG C, concretely 60 DEG C, 61
DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C or 66 DEG C), most preferably 63 DEG C.
The reaction system of the fluorescence ring mediated isothermal duplication of any description above is concretely: 2 μ L, Tris-HCl (pH of template
=8.8) 20mM, KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L,
Bst archaeal dna polymerase 8U, primer HL-F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP
40pmol, primer HL-LF 20pmol, primer HL-LB 20pmol, molecular beacon probe LBP 8pmol.
The reaction condition of the fluorescence ring mediated isothermal duplication of any description above concretely 63 DEG C of isothermal reaction 60min.
Any description above object concretely comma bacillus.When the object is comma bacillus, any of the above
Fluorescence ring mediated isothermal duplication is carried out using primer combination of probe first in the method.
Molecular beacon is introduced into LAMP technology by the present invention, establishes the fluorescence ring mediated isothermal duplication based on molecular beacon
Method, and it is named as MB-LAMP, to realize the direct detection to amplified production, fundamentally solve the side LAMP
The problem of method non-specific amplification, has huge application value and commercial value.
Detailed description of the invention
Fig. 1 is 2 testing result of 1 step 2 of embodiment.
Fig. 2 is the testing result of 1 step 3 of embodiment.
Fig. 3 is the testing result of 1 step 4 of embodiment.
Fig. 4 is the testing result of MB-LAMP reaction in embodiment 2.
Fig. 5 is the testing result of LAMP reaction in embodiment 2.
Fig. 6 is the testing result of LAMP reaction in embodiment 3.
Fig. 7 is the testing result of MB-LAMP reaction in embodiment 3.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
PUC57 carrier: Beijing Tian Yihuiyuan Biotechnology Co., Ltd.
The turbidity of LAMP and the fluorescent value of MB-LAMP are respectively by Loop amp turbidimeter (LA-320c；Eiken
Chemical Co., Ltd.) and CFX Connect Real-Time PCR Detection System (CFX Connect；
BIO-RAD CO., LTD, California, USA) it detects and is analyzed to obtain data.
Comma bacillus positive plasmid: the EcoR of the insertion pUC57 carrier of double chain DNA molecule shown in the sequence 8 by sequence table
Between I restriction enzyme site of V and BamH, obtained recombinant plasmid.
Embodiment 1, the design synthesis of primed probe and detection method optimization
One, the design and synthesis of primer
By the analysis of a large amount of sequences, compare a set of primer combination obtained for identifying comma bacillus.Primer combination packet
Include preceding primer (HL-F3), rear primer (HL-B3), preceding inner primer (HL-FIP), rear inner primer (HL-BIP) and two acceleration primers
(HL-LF,HL-LB).Primer sequence is (5 ' → 3 ') as follows:
HL-F3 (sequence 1 of sequence table): CCTAAATGTAGCAAATTGATTTCCT；
HL-B3 (sequence 2 of sequence table): GTCATTAGGTACTACCGAGG；
HL-FIP (sequence 3 of sequence table): TCCTTTTTTGTAGGGCTATGTTGTTGTTTGTGTGATTTTTGTGTGC；
HL-BIP (sequence 4 of sequence table): ACCATTTGCCTAGCCGTACTACAATAAAGTCACCTTCTTGG；
HL-LF (sequence 5 of sequence table): TGTGTTGCGCGCACAGTA；
HL-LB (sequence 6 of sequence table): TGCAGCCCTACTAGCCGCT.
Two, the design and screening of molecular beacon
1, the matched molecular beacon probe of primer Combination Design obtained for step 1.
Following a few set molecular beacon probes (5 ' → 3 ') are devised altogether:
(1) LBP:AGCGGCTGCAGCCCTACTAGCCGCT；
(2) LBP-1:CAAATATGCAGCCCTACTAGCCGCTCCTGTATTTG；
(3) LBP-2:AGGGCTTGCAGCCCT。
In above-mentioned probe, underscore part is ring neck, and non-underscore part is arm.
Fluorophor FAM is connected in 5 ' ends of each molecular beacon probe, 3 ' ends connect fluorescent quenching group
DABCYL。
2, it using comma bacillus positive plasmid as template, is combined using molecular beacon primer and carries out MB-LAMP reaction.
Molecular beacon primer combination I: 1 molecular beacon of primer combination+step 2 (1) of step 1；
Molecular beacon primer combination II: 1 molecular beacon of primer combination+step 2 (2) of step 1；
Molecular beacon primer combination III: 1 molecular beacon of primer combination+step 2 (3) of step 1；
MB-LAMP reaction system: (25 μ L): template 2 μ L (DNA content 100ng), Tris-HCl (pH=8.8) 20mM,
KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst DNA polymerization
Enzyme 8U, primer HL-F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer
HL-LF 20pmol, primer HL-LB 20pmol, molecular beacon probe is 8pmol.Each molecular beacon primer combination settings
Three repetitions.
MB-LAMP reaction condition: 63 DEG C of isothermal reaction 60min.
As a result as shown in Figure 1.In Fig. 1, abscissa is reaction cycle number, and ordinate is Relative fluorescence units RFU.As a result table
Bright, ring neck length is the probe of 3bp and 23bp during amplified reaction, and fluorescent value swings near baseline and cannot show
The amplification of work.The handle configurations of ring neck structure and 6bp of the optimum structure with 13bp, fluorescent value are reachable during the reaction
To 7000RFU, the progress for detecting reaction can be very good.
Based on the above results, the best molecular beacon probe (5 ' → 3 ') for MB-LAMP is filtered out:
LBP (sequence 7 of sequence table): AGCGGCTGCAGCCCTACTAGCCGCT.
HL-F3, HL-B3, HL-FIP, HL-BIP, HL-LF, HL-LB, LBP form primer combination of probe.
Three, molecular beacon probe content optimizes
Using comma bacillus positive plasmid as template, MB-LAMP reaction is carried out using the primer combination of probe of step 2.
MB-LAMP reaction system: (25 μ L): template 2 μ L (DNA content 100ng), Tris-HCl (pH=8.8) 20mM,
KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst DNA polymerization
Enzyme 8U, primer HL-F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer
HL-LF 20pmol, primer HL-LB 20pmol, molecular beacon probe LBP.
It is different according to the content of molecular beacon probe in the reaction system, it is divided into (each reaction system setting of following system
Repeat three times):
Reaction system 1: the content of molecular beacon probe LBP in the reaction system is 2pmol；
Reaction system 2: the content of molecular beacon probe LBP in the reaction system is 4pmol；
Reaction system 3: the content of molecular beacon probe LBP in the reaction system is 6pmol；
Reaction system 4: the content of molecular beacon probe LBP in the reaction system is 8pmol；
Reaction system 5: the content of molecular beacon probe LBP in the reaction system is 10pmol.
MB-LAMP reaction condition: 63 DEG C of isothermal reaction 60min.
As a result as shown in Figure 2.In Fig. 2, abscissa is reaction cycle number, and ordinate is Relative fluorescence units RFU (103),
Five cluster curves from bottom to top are corresponding in turn to the amplification curve that reaction system 1-5 is obtained.The results show that fluorescence signal
Value rises as the content of molecular beacon rises, but the difference of the 4th and the 5th cluster curve is not obvious, so selection 8pmol
The content final as molecular beacon.
Four, reaction temperature optimizes
Using comma bacillus positive plasmid as template, MB-LAMP reaction is carried out using the primer combination of probe of step 2.
MB-LAMP reaction system: (25 μ L): template 2 μ L (DNA content 100ng), Tris-HCl (pH=8.8) 20mM,
KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst DNA polymerization
Enzyme 8U, primer HL-F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer
HL-LF 20pmol, primer HL-LB 20pmol, molecular beacon probe is 8pmol.
MB-LAMP reaction condition: isothermal reaction 60min.Be arranged different reaction temperatures (60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C,
64 DEG C, 65 DEG C, 66 DEG C), each temperature setting repeats three times.
As a result as shown in Figure 3.In Fig. 3, abscissa is reaction cycle number, and ordinate is Relative fluorescence units RFU.As a result it shows
Show, under the conditions of higher 65,66 DEG C, response curve is unstable, and baseline is not depressed into threshold line or less.At 59 to 64 DEG C
Between, Ct value rises after falling before as the temperature rises, and 63 DEG C of whens correspond to Ct value minimum and reflect efficiency highest.
Embodiment 2, specificity
Sample to be tested are as follows: Bacillus cereus ATCC 14579 (bacillus cereus), Pseudomonas
Aeruginosa ATCC 15692 (Pseudomonas aeruginosa), Enterococcusfaecium ATCC 6569 (enterococcus faecium),
Streptococcus dysgalactiae ATCC 27957 (streptococcus dysgalactiae), Escherichia coli ATCC 25922
(Escherichia coli), Streptococcus uberis ATCC 9927 (streptococcus uberis), Streptococcuspneumonia
ATCC 49619 (streptococcus pneumonia), Salmonella enterica ATCC 14028 (Salmonella enteritidis), Yersinia
Enterocolitica ATCC 9610 (Yersinia enterocolitica), Yersinia enterocolitica ATCC 9610
(Yersinia enterocolitica), Vibrio Parahemolyticus ATCC 17802 (vibrio parahemolyticus),
Staphylococcus aureus ATCC 25923 (aurococcus), Shigellaflexneri ATCC
12022 (Flexner's bacillus), staphylococcus epidermidis ATCC 12228 (staphylococcus epidermis),
11561 (cholera arc of Enterococcus faecalis ATCC 29212 (enterococcus faecalis) and Vibrio cholera ATCC
Bacterium).
1, using the total DNA of sample to be tested as template, MB-LAMP is carried out using the primer combination of probe of 1 step 2 of embodiment
Reaction.
MB-LAMP reaction system (25 μ L): template 2 μ L (DNA content 100ng), Tris-HCl (pH=8.8) 20mM,
KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst DNA polymerization
Enzyme 8U, primer HL-F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer
HL-LF 20pmol, primer HL-LB 20pmol, molecular beacon probe LBP 8pmol.
MB-LAMP reaction condition: 63 DEG C of isothermal reaction 60min.
As a result as shown in Figure 4.In Fig. 4, abscissa is reaction cycle number, and ordinate is Relative fluorescence units RFU.As a result table
Bright, other than comma bacillus can amplify amplification curve, remaining bacterial strain does not obtain amplification curve, it is shown that special well
Property.
2, using the total DNA of sample to be tested as template, LAMP reaction is carried out using the primer combination of 1 step 1 of embodiment.
LAMP reaction system (25 μ L): template 2 μ L (DNA content 100ng), Tris-HCl (pH=8.8) 20mM, KCl
10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst archaeal dna polymerase
8U, primer HL-F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer HL-
LF 20pmol, primer HL-LB 20pmol.
LAMP reaction condition: 63 DEG C of isothermal reaction 60min.
As a result as shown in Figure 5.The result shows that the later period will appear a certain amount of non-specific using common LAMP reaction detection
Amplification carrys out interference experiment result.
Show that the common LAMP reaction detection later period will appear a certain amount of non-specific amplification to interfere reality based on the above results
It tests as a result, and MB-LAMP reaction will not be influenced by non-specific amplification product.
Embodiment 3, sensibility
1, with 10 times of gradient dilution comma bacillus positive plasmids of sterile water, each dilution is obtained.
2, it is anti-to carry out LAMP using the step of embodiment 1 one primer combination as template for each dilution obtained using step 1
It answers.
LAMP reaction system (25 μ L): template 2 μ L, Tris-HCl (pH=8.8) 20mM, KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst archaeal dna polymerase 8U, primer HL-
F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer HL-LF 20pmol,
Primer HL-LB 20pmol, molecular beacon probe LBP 8pmol.
LAMP reaction condition: 63 DEG C of isothermal reaction 60min.
Since the dilution of use is different, following different reaction system is formed:
In reaction system L1, the initial content of Plasmid DNA are as follows: 20ng；
In reaction system L2, the initial content of Plasmid DNA are as follows: 2ng；
In reaction system L3, the initial content of Plasmid DNA are as follows: 200pg；
In reaction system L4, the initial content of Plasmid DNA are as follows: 20pg；
In reaction system L5, the initial content of Plasmid DNA are as follows: 2pg；
In reaction system L6, the initial content of Plasmid DNA are as follows: 200fg；
In reaction system L7, the initial content of Plasmid DNA are as follows: 20fg；
In reaction system L8, the initial content of Plasmid DNA are as follows: 2fg；
In reaction system L9, the initial content of Plasmid DNA are as follows: 0.2fg；
In reaction system L10, Plasmid DNA is substituted with isometric distilled water.
As a result as shown in Figure 6.In Fig. 6, abscissa is time (minute), and ordinate is turbidity (650nm).The result shows that
The lowest detection lower limit of LAMP reaction is 2fg.
3, each dilution obtained using step 1 is carried out as template using the step of embodiment 1 two primer combination of probe
MB-LAMP reaction.
MB-LAMP reaction system: (25 μ L): template 2 μ L, Tris-HCl (pH=8.8) 20mM, KCl 10mM, (NH4)2SO410mM, glycine betaine 0.8M, MgSO48mM, dNTP1.4mM, Tween200.25 μ L, Bst archaeal dna polymerase 8U, primer HL-
F35pmol, primer HL-B35pmol, primer HL-FIP 40pmol, primer HL-BIP 40pmol, primer HL-LF 20pmol,
Primer HL-LB 20pmol, molecular beacon probe LBP 8pmol.
MB-LAMP reaction condition: 63 DEG C of isothermal reaction 60min.
Since the dilution of use is different, following different reaction system is formed:
In reaction system M1, the initial content of Plasmid DNA are as follows: 20ng；
In reaction system M2, the initial content of Plasmid DNA are as follows: 2ng；
In reaction system M3, the initial content of Plasmid DNA are as follows: 200pg；
In reaction system M4, the initial content of Plasmid DNA are as follows: 20pg；
In reaction system M5, the initial content of Plasmid DNA are as follows: 2pg；
In reaction system M6, the initial content of Plasmid DNA are as follows: 200fg；
In reaction system M7, the initial content of Plasmid DNA are as follows: 20fg；
In reaction system M8, the initial content of Plasmid DNA are as follows: 2fg；
In reaction system M9, the initial content of Plasmid DNA are as follows: 0.2fg；
In reaction system M10, Plasmid DNA is substituted with isometric distilled water.
As a result as shown in Figure 7.In Fig. 7, abscissa is reaction cycle number, and ordinate is Relative fluorescence units RFU.In Fig. 7
Be corresponding in turn to the amplification curve that reaction system M1-M9 is obtained from left to right, ct value is followed successively by 26,29,33,37,40,44,48,
52, reaction system M9 and M10 do not obtain amplification curve.The result shows that the lowest detection lower limit of MB-LAMP reaction is similarly 2fg.
Sequence table
<110>Diseases Preventing and Controlling Institute
<120>a kind of fluorescence ring mediated isothermal amplification method based on molecular beacon
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cctaaatgta gcaaattgat ttcct 25
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtcattaggt actaccgagg 20
<210> 3
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tccttttttg tagggctatg ttgttgtttg tgtgattttt gtgtgc 46
<210> 4
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
accatttgcc tagccgtact acaataaagt caccttcttg g 41
<210> 5
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgtgttgcgc gcacagta 18
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tgcagcccta ctagccgct 19
<210> 7
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
agcggctgca gccctactag ccgct 25
<210> 8
<211> 1031
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
agagtattga agatgttgca aaagtgtagc aagttgcaaa cacattatta atgtgcctaa 60
atgtagcaaa ttgatttcct acaagtttgt gtgatttttg tgtgctactg tgcgcgcaac 120
acaaagataa caacatagcc ctacaaaaaa ggaaaacgtc atgaaacaaa ccatttgcct 180
agccgtactt gcagccctac tagccgctcc tgtatttgct caccaagaag gtgactttat 240
tgtgcgcgcg ggtattgcct cggtagtacc taatgacagt agcgataaag tgttaaacac 300
tcaaagtgag ttggcagtta atagcaatac ccacttaggg ttaacgcttg gctatatgtt 360
tactgacaac atcagttttg aagtcctcgc tcgtacgcca ttttcacata agatttctac 420
ctctggtggt gagttaggta gccttggtga tattggtgaa acaaaacatt tgccacctac 480
ctttatggtc caatactact ttggtgaagc taattcgaca aaccgtccat atgttggtgc 540
gggtttgaat tacaccactt tctttgatga aagctttaat agtacgggta ctaataatgc 600
attgagtgat ttaaaactgg acgactcatg gggacttgct gctaacgttg gctttgatta 660
tatgctcaat gatagctggt tcctcaacgc ttatgtgtgg tatgccaata ttgaaacaac 720
ggcaacctac aaagcaggtg cagatgccaa atccacggat gttgaaatca atccttgggt 780
atttatcatc gcgggtggtt ataagttcta acgccctatt tcgaaaataa agccgcgaaa 840
tcgctttatt tttttgtggc ctcgatttca tcatttttag cgtagtgatt gtttctaacg 900
taatctctat tcagagcctg agacgtacgc taatccctgt tgatactgat aaataggtgt 960
caatttaagg cgcaaccata tcgtaagatt tatctttatg atttctttct tattctggct 1020
tcagatcagg t 1031
Claims (9)
1. a kind of primer combination of probe for fluorescence ring mediated isothermal duplication, by LAMP primer composition and molecular beacon probe
LBP composition；The LAMP primer composition is by preceding primer F3, rear primer B3, preceding inner primer FIP, rear inner primer BIP and accelerates primer
LB composition；
To accelerate primer LB people is to be divided into LB-a section and LB-b section；Molecular beacon probe LBP from upstream to downstream successively
It include: LBP-b1 section, LBP-a section and LBP-b2 section；The nucleotide sequence of LBP-a section and the nucleotide of LB-a section
Sequence is identical；LBP-b1 section and LBP-b2 section reverse complemental；The nucleotide sequence of LBP-b1 section or LBP-b2 section with
The nucleotide sequence of LB-b section is identical；
The molecular beacon probe LBP, an end are connected with fluorophor, another end is connected with fluorescent quenching group.
2. primer combination of probe as described in claim 1, it is characterised in that: the LBP-b1 section is made of 6 nucleotide；
The LBP-a section is made of 12-13 nucleotide.
3. primer combination of probe as claimed in claim 2, it is characterised in that:
The preceding primer F3 is following (a1) or (a2)；
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1 identical
The DNA molecular of function；
Primer B3 is following (a3) or (a4) after described；
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2 identical
The DNA molecular of function；
The preceding inner primer FIP is following (a5) or (a6):
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) have by sequence 3 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 3 identical
The DNA molecular of function；
Inner primer BIP is following (a7) or (a8) after described:
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) have by sequence 4 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 4 identical
The DNA molecular of function；
The acceleration primer LF is following (a9) or (a10):
(a9) single strand dna shown in the sequence 5 of sequence table；
(a10) have by sequence 5 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 5 identical
The DNA molecular of function；
The acceleration primer LB is following (a11) or (a12):
(a11) single strand dna shown in the sequence 6 of sequence table；
(a12) have by sequence 6 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 6 identical
The DNA molecular of function；
The molecular beacon probe LBP is following (b1) or (b2):
(b1) single strand dna shown in the sequence 7 of sequence table；
(b2) have by sequence 7 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 7 identical
The DNA molecular of function.
4. the application of primer combination of probe any one of claims 1 to 3 in reagent preparation box；The function of the kit
Can be (c1) or (c2):
(c1) whether detection sample to be tested is object；
(c2) detect in sample to be tested whether contain object.
5. containing the kit of primer combination of probe any one of claims 1 to 3；The function of the kit is (c1)
Or (c2):
(c1) whether detection sample to be tested is object；
(c2) detect in sample to be tested whether contain object.
6. the preparation method of kit described in claim 5 includes the steps that individually packing each primed probe.
7. a kind of detection sample to be tested whether be object method, include the following steps: with the genomic DNA of sample to be tested or
CDNA is template, carries out fluorescence ring mediated isothermal duplication, prison using primer combination of probe any one of claims 1 to 3
Fluorescence signal is surveyed, if showing that positive amplification curve, sample to be tested are or candidate is object, if not showing positive amplification song
Line, sample to be tested are or candidate is non-targeted object.
8. in a kind of detection sample to be tested whether the method containing object, include the following steps: with the total DNA of sample to be tested or
The cDNA that the total serum IgE reverse transcription of sample to be tested obtains is template, using primed probe group any one of claims 1 to 3
It closes and carries out fluorescence ring mediated isothermal duplication, monitor fluorescence signal, if display positive amplification curve, sample to be tested contain or doubtful
Containing object, if not showing that positive amplification curve, sample to be tested do not contain or doubtful without containing object.
9. method as claimed in claim 7 or 8, it is characterised in that: the object is comma bacillus；It is used in the method
Primer combination of probe described in claim 3 carries out fluorescence ring mediated isothermal duplication.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481396A (en) * | 2020-10-28 | 2021-03-12 | 广州迪澳生物科技有限公司 | Kit for detecting rifampin resistance of mycobacterium tuberculosis based on constant temperature method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153332A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting cholera vibrio |
| US20090253183A1 (en) * | 2008-04-03 | 2009-10-08 | Jian Han | Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets |
| CN101892293A (en) * | 2009-05-22 | 2010-11-24 | 深圳出入境检验检疫局食品检验检疫技术中心 | Technology for detecting comma bacillus mdh genes based on loop-mediated isothermal amplification (LAMP) and detection kit thereof |
| CN102888457A (en) * | 2012-09-25 | 2013-01-23 | 江阴天瑞生物科技有限公司 | Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit |
| CN104928357A (en) * | 2014-12-10 | 2015-09-23 | 上海市计量测试技术研究院 | Polynucleotide, method and kit for detecting pathotype vibrio cholerae |
| CN108034700A (en) * | 2018-01-24 | 2018-05-15 | 中国人民解放军军事科学院军事医学研究院 | A kind of isothermal amplification detection method of molecular beacon mediation and its application |
| WO2018089943A1 (en) * | 2016-11-10 | 2018-05-17 | Slipchip Corporation | Probe detection of loop-mediated amplification products |
-
2018
- 2018-09-13 CN CN201811067779.3A patent/CN109055500A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153332A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting cholera vibrio |
| US20090253183A1 (en) * | 2008-04-03 | 2009-10-08 | Jian Han | Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets |
| CN101892293A (en) * | 2009-05-22 | 2010-11-24 | 深圳出入境检验检疫局食品检验检疫技术中心 | Technology for detecting comma bacillus mdh genes based on loop-mediated isothermal amplification (LAMP) and detection kit thereof |
| CN102888457A (en) * | 2012-09-25 | 2013-01-23 | 江阴天瑞生物科技有限公司 | Detection technology of molecular beacon strand displacement isothermal amplifying gene chip and kit |
| CN104928357A (en) * | 2014-12-10 | 2015-09-23 | 上海市计量测试技术研究院 | Polynucleotide, method and kit for detecting pathotype vibrio cholerae |
| WO2018089943A1 (en) * | 2016-11-10 | 2018-05-17 | Slipchip Corporation | Probe detection of loop-mediated amplification products |
| CN108034700A (en) * | 2018-01-24 | 2018-05-15 | 中国人民解放军军事科学院军事医学研究院 | A kind of isothermal amplification detection method of molecular beacon mediation and its application |
Non-Patent Citations (2)
| Title |
|---|
| 朱水芳等: "《实时荧光聚合酶链反应PCR检测技术》", 31 July 2003 * |
| 柯雪梅等: "霍乱弧菌LAMP快速检测方法的建立及应用", 《南方医科大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481396A (en) * | 2020-10-28 | 2021-03-12 | 广州迪澳生物科技有限公司 | Kit for detecting rifampin resistance of mycobacterium tuberculosis based on constant temperature method |
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