CN109797195A - Freeze-draw method rapid detection method and its application based on analysis PCR by-product pyrophosphoric acid - Google Patents
Freeze-draw method rapid detection method and its application based on analysis PCR by-product pyrophosphoric acid Download PDFInfo
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Abstract
The invention discloses a kind of freeze-draw method rapid detection method based on analysis PCR by-product pyrophosphoric acid and its applications, and this method comprises the following steps: extracting sample genome and react for PCR；(2) it designs the PCR primer contained at least one and carries out PCR amplification with the thio deoxyadenosine triphosphate substitution deoxyadenosine triphosphate of α-；(3) pyrophosphoric acid in pcr amplification product is analyzed；(4) determine whether PCR reaction occurs based on the analysis results, detect to contain DNA content to be measured in sample.The method of the present invention can go out in sample whether limit containing sample DNA and its content, its detection up to several copies with accurate detection；The determination method high sensitivity, detection is fast, and operating process is simply easy to implement, and can implement quickly detection to large sample freeze-draw method, the quick detection that can be used for the microorganism of the industries such as food, health can be used for other nucleic acid sequence analysis with specific PCR reaction.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of specific PCR production based on analysis PCR by-product pyrophosphoric acid
Object rapid detection method and its application, suitable for specific DNA fragments such as food safety, drinking water safety monitoring, clinical samples
Quickly analysis and identification.
Background technique
Round pcr is a kind of for amplifying the Protocols in Molecular Biology for expanding specific DNA fragmentation, can be by micro DNA
It is significantly increased, by the quickly method analysis such as simple electrophoresis, then can clearly judge whether PCR reaction is sent out by electrophoretic band
It whether there is specific DNA fragmentation in raw, judgement sample.This round pcr (SNP, is dashed forward in analysis such as mononucleotide variation
Become, insertion, seven mistakes) analysis (such as rolling circle amplification), invasive organism is (such as: salmonella, staphylococcus, streptococcus, secondary haemolysis
Property vibrios, proteus, Shigella, avian influenza virus, Aspergillus flavus and virus, foot and mouth disease virus etc.) etc. obtain in detection
It is widely applied.However, the object of all analyses is the DNA generated in PCR reaction in the detection technique of existing based on PCR
Sequence, theoretical amount are the 2 of sample amplifying nucleic acid copy numberN-1(N is PCR cycle number), if simple using electrophoresis etc., quick
Method analysis, then sample amplifying nucleic acid copy number need to reach it is hundreds of could pass through electrophoretic bands clearly judge PCR react be
No generation can not be implemented effectively to detect to low abundance sample.Such as cause human diseases in low copy number and that poisons by food causes a disease
Property microorganism need to detect using traditional cultivation, it is complex for operation step, time-consuming, be not able to satisfy and be quickly detected on diagnosis
Demand.
In fact, one nucleotide of every synthesis, PCR primer will extend a bases longs, simultaneously during PCR
Also a by-product pyrophosphoric acid molecule can be supervened.It is assumed that there are a DNA moleculars in sample, if every primer exists
Extend 100 bases during PCR, to generate 200 pyrophosphoric acid molecules in a PCR process in a pair of of DNA profiling；Namely
During saying PCR, the pyrophosphoric acid molecule amount of generation is generally the hundred times of DNA.Therefore, the PCR recycled by tens is anti-
It answers, the theoretical amount of pyrophosphoric acid can achieve 10-11Mole (such as PCR carries out 36 circulations, then the PCR for synthesizing 100 bases is produced
The pyrophosphoric acid amount that object generates is (2 × 100 × 235)/(6×1023)=1.1 × 10-11Mole；Its concentration can achieve 1.1 × 10-11Mole/25 μ L=0.4mM), it is sufficient to reach the 10 of general chemiluminescence analysis-12Gram detection limit.Therefore, selection analysis PCR pair
Product pyrophosphoric acid, rather than its sensitivity of PCR product DNA fragmentation is bigger, is more suitable for the analysis of low abundance nucleic acid sequence.
Existing very more analysis method is used for the quantitative analysis tech of pyrophosphoric acid, the typical case analyzed using pyrophosphoric acid
Example is burnt sequencing.Its principle is that reaction substrate is 5'- phosphosulfate, fluorescein, in adenosine triphyosphate sulfurylase
Under the action of, pyrophosphoric acid can combine with 5'- phosphosulfate and form adenosine triphyosphate, under the catalysis of luciferase,
The adenosine triphyosphate of generation can combine with fluorescein again and form oxyluciferin, while generate visible light.Pass through inspection
Surveying device can get a detection peak, and the height of peak value and the amount of pyrophosphoric acid are directly proportional.
However, (Xiao Pengfeng, Zhou Dongrui, old using the method that burnt sequencing principle directly analyzes pyrophosphoric acid in PCR by-product
Detection Method of Pathogenic Microorganism Chinese invention patent application number of the tinkling of pieces of jade based on analysis PCR by-product pyrophosphoric acid
201710042607.X), the starting DNA profiling for detecting shigella dysenteriae still needs 25 L × 1.26 × 10 μ-6Ng/ μ L=3.15 ×
10-5Ng (about 104Copy number) more than be just able to achieve effective detection, be not suitable for lower concentration DNA profiling detection.It is main
The reason is that the conventional dNTPs itself being added in PCR system contains the chemiluminescent substance dATP of pyrophosphoric acid, it and pyrophosphoric acid are chemical
Luminous substrate adenosine phosphosulfate (APS) structure is similar, can be decomposed by luciferase, influences on fluorescent strength determining very big.This
Outside, pyrophosphoric acid also can slowly degrade with the time.Therefore, although being greater than a certain amount of sample, phase for starting DNA profiling number
Conclusive influence is not generated for the blank sample without specific DNA profiling, but starting DNA profiling number is less than a certain amount of
Sample, it is possible to conclusive influence is generated on detection, that is, can not achieve detection.
Summary of the invention
Goal of the invention: for it is existing based on analysis PCR by-product pyrophosphoric acid freeze-draw method rapid detection method in deposit
Detection limit problem, the present invention provides a kind of quick side of detection of freeze-draw method based on analysis PCR by-product pyrophosphoric acid
Method, specific dna sequence of this method according to pattern detection, the PCR primer of design, by using the thio deoxyadenosine triphosphate of α-
The genomic PCR amplification of (dATP α S) substitution deoxyadenosine triphosphate (dATP) progress simultaneously analyzes the total coke generated in PCR amplification
Phosphoric acid determine PCR reaction whether occur with occur number, thus derive whether contain the specific genome and its DNA it is dense
Degree.The determination method high sensitivity, fastly, operating process is simple for detection, is easy to implement, and can implement quickly to divide to large sample
Analysis, while can solve DNA profiling copy number low the problem of cannot effectively detecting, by directly analyzing pyrophosphoric acid in PCR by-product
Realize the detection to low copy number DNA template, method of the invention can reduce or eliminate what dATP in blank sample was generated
Background value, enough quick, low costs realize the detection to low copy number DNA template by pyrophosphoric acid in directly analysis PCR by-product.
The invention also discloses the applications of the freeze-draw method rapid detection method based on analysis PCR by-product pyrophosphoric acid.
Technical solution: to achieve the goals above, a kind of based on analysis PCR by-product pyrophosphoric acid as described in the present invention
Freeze-draw method rapid detection method, includes the following steps:
(1) sample genome is extracted, genome is reacted for PCR；
(2) it designs the PCR primer contained at least one and substitutes three phosphorus of desoxyadenossine with the thio deoxyadenosine triphosphate of α-
Acid carries out PCR amplification；If being one using primer in rolling circle amplification；Conventional amplification is a pair of；There are also other 3-6 primers
The more autogamys of such as isothermal cause amplification mode；
(3) by-product pyrophosphoric acid in analytical procedure (2) PCR amplification；
(4) determine whether PCR reaction occurs based on the analysis results, and detect to contain DNA content to be measured in sample.
Wherein, the freeze-draw method rapid detection method based on analysis PCR by-product pyrophosphoric acid, feature exist
In the specific PCR refers to that primer only expands specific nucleic acid sequence fragments, does not expand other sequence fragments.
Wherein, genome described in step (1) is reacted directly or through after processing for PCR, described to be by processing
Refer to that genome passes through the biochemical reactions such as digestion, annulation.
Further, step (2) PCR primer contained at least one that designs is for specific nucleic acid in genome
Sequence fragment (such as conventional amplification) or (the more autogamys of such as rolling circle amplification, isothermal cause amplification for artificial synthesized sequence fragment
Deng) be designed, the specific nucleic acid sequence fragments refer to uniquely having, not having in other biologies, simultaneously for certain biology
And the nucleic acid sequence information that can be obtained by common data base；The artificial synthesized sequence fragment refers to by certain life
Object genome person after treatment, building cause the new DNA profiling of amplification etc. for the more autogamys of rolling circle amplification, isothermal.
Preferably, PCR amplification described in step (2) includes the isothermal or alternating temperature PCR of single, double or more primers.
Wherein, step (2) is described carries out PCR amplification with α-thio deoxyadenosine triphosphate substitution deoxyadenosine triphosphate and is
Substitute deoxyadenosine triphosphate dATP with the thio deoxyadenosine triphosphate dATP α S of α-, excess-three kind monomer be routine dNTPs into
Row, i.e. dGTP, dTTP and dCTP.
Wherein, pyrophosphoric acid is by pyrophosphoric acid chemiluminescence analysis, directly in step (3) the analysis PCR amplification by-product
The pyrophosphoric acid in analysis PCR product is connect to realize.
The method of step (3) the pyrophosphoric acid chemiluminescence analysis is implemented or is led to by using pyrophosphoric acid detection kit
The chemical illuminating reagent of preparation is crossed to implement；The pyrophosphoric acid detection kit is that the pyrophosphoric acid of SIGMA-ALDRICH company is examined
Test agent box；The chemical illuminating reagent main ingredient of the preparation are as follows: 0.01~0.5M Tris-acetate (pH 7.7), 10
~50mM EDTA, 1~5mM Mg (Ac)2, 1~5mM dithiothreitol (DTT) (DTT), 10~50mM 5'- phosphosulfate (APS),
0.2~1.0mg/mL polyvinylpyrrolidone (PVP), 2~10mM fluorescein (D-luciferin), 50~800mM fluorescein
Enzyme.
Preferably, in the pyrophosphoric acid chemiluminescence analysis optical signal detection photodiode or Charged Couple
Element CCD；It can individually detect, it can also be with parallel detection.
Wherein, determine whether PCR reaction occurs described in step (4) based on the analysis results, be the pyrophosphoric acid by setting
The CV value of analysis judges that CV value is usually set to 3 times of blank sample: pyrophosphoric acid analyte signal intensity is greater than CV value and is determined as
, be corresponding to it to be containing DNA to be measured in sample；Less than CV value be determined as without, be corresponding to it for be free from sample to
Survey DNA；
Further, detected described in step (4) in sample containing DNA content to be measured refer to first pass through it is a series of
Know that concentration DNA profiling by step (1)-(4), after establishing the relation curve of concentration and signal strength, then analyzes what sample obtained
Signal strength acquires the specific concentration of the DNA from relation curve.
Relative in pyrophosphoric acid chemiluminescence analysis, influence of the dATP to luminous intensity, (S atom is instead of dATP by dATP α S
O atom on molecule alpha P=O) replace influence of the dATP to luminous intensity under natural conditions to want much less.The present invention is directed to existing PCR
Influence of the dATP to pyrophosphorylation luminesceence analysis in product substitutes deoxidation using the thio deoxyadenosine triphosphate of α-(dATP α S)
Adenosine triphosphate (dATP) carries out PCR amplification, so that dATP is to pyrophosphorylation luminesceence analysis in reduction or elimination PCR product
Influence, realize based on analysis PCR by-product pyrophosphoric acid PCR product rapid detection method, quickly, low cost for food pacify
Entirely, the Rapid identification of the specific DNA fragments such as drinking water safety monitoring, clinical sample.
The utility model has the advantages that compared with prior art, the present invention has the advantage that
1, the present invention provides a kind of rapid detection method for freeze-draw method, that is, passes through the coke in detection PCR by-product
Phosphoric acid can determine in sample whether contain DNA to be measured and its DNA content.
2, the PCR reaction that the present invention is carried out using dATP α S substitution dATP drastically reduces pyrophosphoric acid detection chemistry hair
The background value of light detection, detected value up to several DNA copies (low copy number), even single DNA copy (but due to sample not
Can guarantee can get DNA), detection sensitivity is greatly improved.
3, the present invention can be completed point using pyrophosphoric acid content in directly detection PCR product after the completion of PCR, in 3 minutes
Analysis, detection speed are faster than traditional detection DNA molecular method.
4, method of the invention is suitble to the detection of the room temperature, alternating temperature amplified production of existing single, double, more primer.
5, method of the invention can implement large sample quickly to analyze, and accurate quantification and qualification can be used for
Detection.
6, the present invention can use easy optical detection apparatus, and operating process is simple, be easy to implement.
7, detection method of the invention can effectively apply to food safety, drinking water safety monitoring, clinical sample diagnosis
Etc. in the Rapid identification of specific DNA fragments, it can be achieved that the quick detection of the microorganism of the industries such as food, health, can be used for
Other nucleic acid sequence analysis with specific PCR reaction.
Detailed description of the invention
Fig. 1 is the light signal strength standard of PCR product under different salmonella DNA template concentrations in the embodiment of the present invention 1
Curve graph.
Specific embodiment
The present invention will be further described with attached drawing with reference to embodiments.
Embodiment 1
The detection of salmonella in chicken
1, genomic DNA sample preparation: the bacterium on chicken surface is rinsed using PBS (pH=7.2), is then used
DNA of bacteria extracts kit (QIAamp fast DNA stool mini kit, Qiagen) mentions the DNA of bacteria rinsed
It takes out, cryo-conservation is spare.
2, PCR primer design is reacted with PCR
For salmonella nucleic acids characteristic sequence fragment, salmonella invA gene is the base of coding secretion virulence protein
Cause, it is closely related with the bacteria pathogenic, it is that Salmonella is distinctive, according to GenBank accession no.M76445.1
Design forward primer and reverse primer:
SEQ ID NO.1 is positive primers F: 5 '-CATCTGTCTCGCCTCCTG-3 ',
SEQ ID NO.2 is reverse primer R:5 '-GGGCCATTTGTCTACTCATA-3 '；
(in GeneBank database retrieve amplification gene sequence, be submitted in primer-design software Primer5.0 into
Row design of primers, property can be carried out homologous by NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)
Sequence alignment), it is desirable that primer is strong, nothing or less primer dimer, miscellaneous band, hangover etc..
The primer of design is synthesized and does following PCR reaction:
(1) measurement of standard curve: the PCR amplification system of 25 μ L includes: salmonella gene group DNA copy number is respectively
1.0×106、1.0×105、1.0×104、1.0×103、1.0×102, 10,5,0.2mM dNTPs (wherein dATP α S substitute
DATP, remaining is conventional monomeric) the PCR reaction that carries out, 0.8 μM of forward primer and 0.8 μM of reverse primer, 2U Taq DNA polymerization
Enzyme (stoste of Taq archaeal dna polymerase is 1000U/mL), 1 × amplification buffer, 1.5mM MgCl2.Amplification condition: 94 DEG C of pre- changes
Property 5 minutes, 35 thermal cycles (94 DEG C of 30 seconds~52 DEG C of denaturation, 30 seconds~72 DEG C of annealing extend 30 seconds), last 72 DEG C extend 5 points
Clock.
(2) detection of actual sample: the PCR amplification system of 25 μ L includes: 200ng genomic DNA, 0.2mM dNTPs (its
Middle dATP α S substitutes dATP, remaining is conventional monomeric), 0.8 μM of forward primer and 0.8 μM of reverse primer, 2U Taq DNA polymerization
Enzyme, 1 × amplification buffer, 1.5mM MgCl2.Amplification condition: 94 DEG C initial denaturation 5 minutes, (94 DEG C are denaturalized 30 seconds for 35 thermal cycles
~52 DEG C 30 seconds~72 DEG C of annealing extend 30 seconds), last 72 DEG C extend 5 minutes.
3, PCR product detects
(1) chemical luminous system prepares
Using following homemade formula reaction system (S-mix): 40 μ L chemical luminous system (S-mix) solution include:
0.01M Tris-acetate(pH 7.7),20mM EDTA,1mM Mg(Ac)2, 1mM dithiothreitol (DTT) (DTT), 10mM adenosine
Acyl sulfate (APS), 0.4mg/mL polyvinylpyrrolidone (PVP), 4mM fluorescein (D-luciferin), 800mM luciferase.
(2) PCR product detects
A. Weak light investigating instrument, 25 DEG C of setting temperature and high-voltage value 800V are opened.
B. prepare 1.5mL centrifuge tube, 40 μ L chemical luminous system (S-mix) solution are added.
C. it checks whether the shutter of Weak light investigating instrument closes, the lid of sample room is opened under shutter close, will be centrifuged
Pipe is put into sample room, covers upper cover, opens shutter, measures the background signal of chemical luminous system solution.
D. it closes and opens upper cover behind the door fastly, take out centrifuge tube.The PCR of 1 μ L is added in chemical luminous system solution is added
Amplified production, oscillation are uniformly mixed.It is then placed in sample room, opens shutter, measurement obtains the optical signal value of pcr amplification product.
The data that record measurement obtains.
E. after measuring, shutter, measuring instrument are closed.
F. according to blank sample measured value, CV value (as blank sample measured value is determined with three times blank sample measured value
Three times, in blank sample be free of salmonella DNA, other agents useful for same and analysis method are identical as sample), by institute's sample
Fluorescence signal value and CV value evolve compared with, signal strength is greater than CV value and is judged to having, be corresponding to it to be to contain this in sample
Microorganism, less than CV value be determined as without, be corresponding to it to be free from microorganism in sample.
(3) result judges
A. measurement result: the measurement result for drawing standard curve and actual sample is shown in Table 1.According to the fluorescence under various concentration
Signal value, it is positive sample that the signal strength for measuring sample, which is greater than 260, indicates to contain the microorganism in the sample.Signal is strong
Degree is negative sample lower than 260, is indicated in the sample without the microorganism.
The light signal strength of PCR product under the different DNA template concentrations of table 1..
B. it the drafting of standard curve: is drawn and is surveyed according to the light signal strength (Y) that known dna template concentrations (C) in table 1 measure
Curve graph is measured, as shown in Figure 1, the fitting equation that fitting obtains is Y=2200X, X is the logarithm (lgC) of DNA concentration, and Y is to survey
The light signal strength obtained subtracts the light signal strength of blank control.
C. actual sample testing result: the present embodiment 1 the signal strength of sample 1 is 4500, according to standard curve (figure
1), it is known that its salmonella DNA concentration copy number detected is about 100；The signal strength of sample 2 is 7500, it is known that it is detected
Salmonella DNA concentration copy number be 2500；Sample 3 and blank control 88 are suitable, are lower than CV value 260, and sand is not detected in expression
Door Salmonella.
Comparative example 1: salmonella contrasting detection method
(1) chemiluminescence detection of the dNTPs monomer PCR by-product pyrophosphoric acid of salmonella gene group DNA known to
1, genomic DNA sample preparation: the bacterium on chicken surface is rinsed using PBS (pH=7.2), is then used
The DNA of bacteria rinsed is extracted and determines the concentration of genomic DNA by DNA of bacteria extracts kit.
2, PCR primer design is reacted with PCR
For salmonella nucleic acids characteristic sequence fragment, salmonella invA gene is the base of coding secretion virulence protein
Cause, it is closely related with the bacteria pathogenic, it is that Salmonella is distinctive, according to GenBank accession no.M76445.1
Design forward primer and reverse primer:
SEQ ID NO.1 is positive primers F: 5 '-CATCTGTCTCGCCTCCTG-3 ',
SEQ ID NO.2 is reverse primer R:5 '-GGGCCATTTGTCTACTCATA-3 '；
(in GeneBank database retrieve amplification gene sequence, be submitted in primer-design software Primer5.0 into
Row design of primers, property can be carried out homologous by NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/)
Sequence alignment), it is desirable that primer is strong, and nothing or less primer dimer are misquoted.
The primer of design is synthesized and does following PCR reaction (it is blank control that DNA profiling, which is not added):
The PCR amplification system of 25 μ L includes: salmonella gene group DNA copy number is respectively 1.0 × 106、1.0×105、
1.0×104、1.0×103、1.0×102, 5, the PCR reaction that carries out of 10,0.2mM dNTPs, 0.8 μM of forward primer and 0.8 μM
Reverse primer, 2U Taq archaeal dna polymerase, 1 × amplification buffer, 1.5mM MgCl2.Amplification condition: 94 DEG C initial denaturation 5 minutes,
35 thermal cycles (94 DEG C of 30 seconds~52 DEG C of denaturation, 30 seconds~72 DEG C of annealing extend 30 seconds), last 72 DEG C extend 5 minutes.
3, PCR product detects
(1) chemical luminous system prepares
Using following homemade formula reaction system (S-mix): 0.01M Tris-acetate (pH 7.7), 20mM
EDTA,1mM Mg(Ac)2, 1mM dithiothreitol (DTT) (DTT), 10mM adenosine phosphosulfate (APS), 0.4mg/mL polyvinylpyrrolidine
Ketone (PVP), 4mM fluorescein (D-luciferin), 800mM luciferase.
(2) PCR product detects
A. Weak light investigating instrument is opened, temperature and high-voltage value are set.
B. prepare 1.5mL centrifuge tube, 40 μ L chemical luminous system (S-mix) solution are added.
C. it checks whether the shutter of Weak light investigating instrument closes, the lid of sample room is opened under shutter close, will be centrifuged
Pipe is put into sample room, covers upper cover, opens shutter, measures the background signal of chemical luminous system solution.
D. it closes and opens upper cover behind the door fastly, take out centrifuge tube.The PCR of 1 μ L is added in chemical luminous system solution is added
Amplified production, oscillation are uniformly mixed.It is then placed in sample room, opens shutter, measurement obtains the optical signal value of pcr amplification product.
The data that record measurement obtains.
E. after measuring, shutter, measuring instrument are closed.
(3) measurement result: table 2 gives the chemistry of dNTPs monomer PCR by-product pyrophosphoric acid under different DNA template concentrations
Shine testing result.As can be seen from the table, only DNA content is greater than 104The light signal strength for copying numerical example could be with sky
White sample has an apparent difference, and DNA content 103And its light signal strength and blank sample of following copy numerical example do not have area
Not, it can not achieve effective detection.The comparison present invention substitutes dATP using dATP α S, remaining is burnt for the PCR by-product of conventional monomeric
The analysis of phosphoric acid not only remains the characteristics of detection is fast, and operating process is simple, easy implementation, and detection limit can achieve several
DNA copy number substantially increases the sensitivity of detection, has opened up extensively application range.
The chemiluminescence detection result of dNTPs monomer PCR by-product pyrophosphoric acid under the different DNA template concentrations of table 2.
Comparative example 2
Salmonella cultivates detection method national standard GB/T 4789.4-2003
1, it prepares the pre- enrichment liquid of BPW: weighing BPW culture medium 20.1g, distilled water 1L is added, agitating and heating is boiled to complete
Dissolution, is dispensed into each conical flask with 90mL/ bottles, and in 121 DEG C of sterilizing 15min, be cooled to room temperature.
2, after culture medium is cooled to room temperature, culture medium and entire room are carried out ultraviolet sterilization 15 minutes, after the 60min that turns off the light
It uses.
3, increase bacterium in advance: weighing the conical flask of chicken meat sample 10g to the culture medium containing BPW, and close the lid in time, under 150rpm
10min is vibrated, in 36 DEG C of ± 1 DEG C of culture 18h or so.
4, it prepares TTB enrichment liquid: taking TTB enrichment liquid basis 93.6g, distilled water 1L is added, agitating and heating is boiled to complete
Dissolution, is sub-packed in conical flask, every bottle of 100mL.Conical flask is put into autoclave, 121 DEG C of sterilizing 20min；It is cooled to 30 DEG C, often
TTB matched reagent iodine solution and brilliant green one each (preceding 75% alcohol swab disinfection cillin bottle of unlatching are added in 100mL basal medium
Surface), it is uniformly mixed.
5, increase bacterium
6, it is inoculated with and cultivates: gently shaking the sample mixture cultivated, pipette 1mL, transferred species is in 9mL TTB enrichment liquid
It is interior, in 42 DEG C of ± 1 DEG C of culture 18h~for 24 hours.
7, observation culture phenomenon.
8, salmonella color culture medium plate is prepared.
9, dry powder 4.75g in bottle is taken, is dissolved with 100mL distilled water, can be scaled up or reduce.
10,100 DEG C are mixed and heated to, is heated with stirring to and is completely dissolved, is cooled to 50 DEG C of inverted plates.
11, sample setting-out or rubbing method inoculation, 37 DEG C are cultivated 24 hours, purify for 3 generations.
12, colony colour is observed.
13, in conjunction with serological test, biochemical test, 16SrDNA test is identified.
By the comparative analysis of actual sample culture detection method, the PCR of the invention based on analysis PCR by-product pyrophosphoric acid
Product rapid detection method is consistent with culture detection method result for Detection Method of Pathogenic Microorganism.
Culture detection method is a kind of stringent, accurate microorganism detection method, but this method operating process is complicated, time-consuming
It is long, be not suitable for the quick detection of microorganism.Therefore, the present invention is based on the freeze-draw method of analysis PCR by-product pyrophosphoric acid is quick
Detection method high sensitivity, detection are fast, and operating process is simple, are easy to implement, and can implement fast qualitative, quantitative point to large sample
Analysis is very suitable to the microorganism of the industries such as food, health and the quick detection of other specific PCRs reaction.
Embodiment 2
Embodiment 2 uses and the identical method of embodiment 1, the difference is that pyrophosphoric acid chemiluminometry uses
The pyrophosphoric acid detection kit of SIGMA-ALDRICH company purchase, testing result are similar to Example 1.
Embodiment 3
Rs11053646SNP loci detection method based on analysis rolling circle amplification by-product pyrophosphoric acid
1, DNA sample is extracted and handled: peripheral blood sample is mentioned using traditional protein kinase K and phenol/chloroform extraction process
Take the genomic DNA in peripheral blood.Genomic DNA limitation restriction endonuclease enzyme Ase I (New England Biolabs, United
States it) digests.
2, the connection reaction of ring:
(1) the connection reaction of ring 1: the genome comprising 100ng limitation restriction endonuclease enzyme Ase I digestion in 10 μ L reaction volumes
The artificial synthesized open-circle DNA sequence SEQ ID NO.3:(5'-PO of DNA, 1nM4 3—
AGCCAAGAGAAGTGCGTAACGGTGTGAT ATGAGCGTGATCGTGCCTTGTCATTCGGGAAACTGGGAAAAG-3 '),
2U ligase (Epicentre Technologies), 26mmol/L tris-HCl (pH=8.3), 32.5mmol/L KCl,
13mmol/l MgCl2, 0.65mmol/L NAD, and 0.013%X-100.Then it reacts and is carried out 5 minutes at 95 DEG C,
40 thermal cycles: 95 DEG C of denaturation, 20 seconds~65 DEG C annealing, connection 1min, last 65 DEG C connect 10 minutes.It connects after the reaction was completed,
10U excision enzyme I (New England Biolabs, Ipswich, MA) 37 DEG C of reaction 3h degradations not cyclic open-circle DNA sequence is added
Column, then 80 DEG C of reaction 20min inactivate excision enzyme I.
(2) the connection reaction of ring 2: the genome comprising 100ng limitation restriction endonuclease enzyme Ase I digestion in 10 μ L reaction volumes
The artificial synthesized open-circle DNA sequence SEQ ID NO.4:(5'-PO of DNA, 1nM4 3--AGCCAAGAGAAGTGCGTAACGGTGT
GATATGAGCGTGATCGTGCCTTGTCATTCGGGAAACTGGGAAAAC-3 '), 2U ligase (Epicentre
Technologies), 26mmol/L tris-HCl (pH=8.3), 32.5mmol/L KCl, 13mmol/l MgCl2,
0.65mmol/L NAD and 0.013%X-100.Then it reacts and is carried out 5 minutes at 95 DEG C, 40 thermal cycles: 95
DEG C denaturation 20 seconds~65 DEG C annealing, connection 1min, it is last 65 DEG C connect 10 minutes.After the reaction was completed, 10U excision enzyme is added in connection
The not cyclic open-circle DNA sequence of 37 DEG C of I (New England Biolabs, Ipswich, MA) reaction 3h degradations, then 80 DEG C it is anti-
20min is answered to inactivate excision enzyme I.
3, rolling circle amplification: in 20 μ L reaction volumes (wherein comprising the connection product 0.6mmol/l dNTPs after 1 μ L degradation
DATP α S substitutes dATP, remaining is conventional monomeric), 1 μm of ol/L amplimer SEQ ID NO.5 (5'-
CATACGACGCTCATATCACACCGT-3 '), 4U Bst polymerase (New England Biolabs), 20mmol/L Tris-
HCl(pH 8.8),10mmol/L KCl,10mmol/L(NH4)2SO4With 0.1%Triton X-100.Rolling circle amplification is at 55 DEG C
React 3h.
4, rolling circle amplification product detects
(1) chemical luminous system prepares
Using following homemade formula reaction system (S-mix): 0.01M Tris-acetate (pH 7.7), 20mM
EDTA,1mM Mg(Ac)2, 1mM dithiothreitol (DTT) (DTT), 10mM adenosine phosphosulfate (APS), 0.4mg/mL polyvinylpyrrolidine
Ketone (PVP), 4mM fluorescein (D-luciferin), 800mM luciferase.
(2) rolling circle amplification product detects
A. Weak light investigating instrument is opened, temperature and high-voltage value are set.
B. prepare 1.5mL centrifuge tube, 40 μ L chemical luminous system (S-mix) solution are added.
C. it checks whether the shutter of Weak light investigating instrument closes, the lid of sample room is opened under shutter close, will be centrifuged
Pipe is put into sample room, covers upper cover, opens shutter, measures the background signal of chemical luminous system solution.
D. it closes and opens upper cover behind the door fastly, take out centrifuge tube.The rolling ring of 1 μ L is added in chemical luminous system solution is added
Amplified production, oscillation are uniformly mixed.It is then placed in sample room, opens shutter, measurement obtains the optical signal value of rolling circle amplification product.
The data that record measurement obtains.
E. after measuring, shutter, measuring instrument are closed.
(3) result judges
Compare two rolling circle amplification product by-product pyrophosphoric acid chemiluminescence analysis results: if (1) two amplified productions
Detected value be all larger than three times blank sample value, then the sample be heterozygous；(2) if the detected value of 1 amplified production of ring is big
It is less than three times blank sample value in the detected value of three times blank sample value and 2 amplified production of ring, then the sample is that " G " is homozygous；
(3) if the detected value of 2 amplified production of ring is all larger than the detected value of three times blank sample value and 1 amplified production of ring less than three times
Blank sample value, then the sample is that " C " is homozygous.
Sequence table
<110>Southeast China University
<120>freeze-draw method rapid detection method and its application based on analysis PCR by-product pyrophosphoric acid
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
catctgtctc gcctcctg 18
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gggccatttg tctactcata 20
<210> 3
<211> 70
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agccaagaga agtgcgtaac ggtgtgatat gagcgtgatc gtgccttgtc attcgggaaa 60
ctgggaaaag 70
<210> 4
<211> 70
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
agccaagaga agtgcgtaac ggtgtgatat gagcgtgatc gtgccttgtc attcgggaaa 60
ctgggaaaac 70
<210> 5
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
catacgacgc tcatatcaca ccgt 24
Claims (10)
1. a kind of freeze-draw method rapid detection method based on analysis PCR by-product pyrophosphoric acid, which is characterized in that including such as
Lower step:
(1) sample genome is extracted, genome is reacted for PCR；
(2) PCR primer that contains at least one of design and with the thio deoxyadenosine triphosphate substitution deoxyadenosine triphosphate of α-into
Row PCR amplification；
(3) by-product pyrophosphoric acid in analytical procedure (2) PCR amplification；
(4) determine whether PCR reaction occurs based on the analysis results, and detect to contain DNA content to be measured in sample.
2. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, the specific PCR refers to that primer only expands specific nucleic acid sequence fragments, does not expand other sequence fragments.
3. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, genome described in step (1) is reacted directly or through after processing for PCR, described to refer to base by processing
Because group passes through digestion, annulation.
4. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, step (2) PCR primer contained at least one that designs is for specific nucleic acid sequence fragments in genome
Or be designed for artificial synthesized sequence fragment, the specific nucleic acid sequence fragments refer to unique tool of certain biology
The nucleic acid sequence information for having, not having and can be obtained by common data base in other biologies；The artificial synthesized sequence
Column-slice section refer to by biological genome person after treatment, building for rolling circle amplification or the more autogamys of isothermal cause amplification
New DNA profiling.
5. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, PCR amplification described in step (2) includes the isothermal or alternating temperature PCR of single, double or more primers.
6. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, step (2) is described, and to carry out PCR amplification with α-thio deoxyadenosine triphosphate substitution deoxyadenosine triphosphate be with α-
Thio deoxyadenosine triphosphate dATP α S substitutes deoxyadenosine triphosphate dATP, and excess-three seed nucleus nucleotide monomers are routine dNTPs
Carry out i.e. dGTP, dTTP and dCTP.
7. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, pyrophosphoric acid is preferably by pyrophosphoric acid chemiluminescence analysis in step (3) the analysis PCR amplification by-product, directly
The pyrophosphoric acid in analysis PCR product is connect to realize.
8. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid, step
Suddenly determine whether PCR reaction occurs described in (4) based on the analysis results, sentenced by the CV value of the pyrophosphoric acid analysis of setting
Disconnected: pyrophosphoric acid analyte signal intensity is greater than CV value and is judged to having, be corresponding to it to be containing DNA to be measured in sample；Less than CV
Value is determined as nothing, is corresponding to it to be free from DNA to be measured in sample.
9. the freeze-draw method rapid detection method according to claim 1 based on analysis PCR by-product pyrophosphoric acid,
It is characterized in that, detects to refer to containing DNA content to be measured in sample and first pass through a series of known concentrations described in step (4)
DNA profiling passes through step (1)-(4), and after establishing the relation curve of concentration and signal strength, then to analyze the signal that sample obtains strong
Degree, acquires the specific concentration of the DNA from relation curve.
10. a kind of freeze-draw method rapid detection method described in claim 1 based on analysis PCR by-product pyrophosphoric acid exists
Food safety, drinking water safety monitoring, clinical sample specific DNA fragments Rapid identification in application.
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Application publication date: 20190524 |