CN106701966A - Rapid pathogenic microorganism detection method based on analysis on PCR (Polymerase Chain Reaction) byproduct pyrophosphoric acid - Google Patents
Rapid pathogenic microorganism detection method based on analysis on PCR (Polymerase Chain Reaction) byproduct pyrophosphoric acid Download PDFInfo
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Abstract
The invention discloses a rapid pathogenic microorganism detection method based on analysis on a PCR (Polymerase Chain Reaction) byproduct pyrophosphoric acid. The rapid pathogenic microorganism detection method comprises the following steps of: (1) retrieving pathogenic microorganism characteristic nucleotide sequence fragment; (2) designing a pair of specific PCR primers for the characteristic nucleotide sequence fragment; (3) extracting a microorganism genome of a sample to be detected, and performing PCR amplification by using the specific PCR primer; (4) analyzing pyrophosphoric acid in PCR amplification; and (5) determining whether a PCR is implemented or not and the reaction intensity according to analysis results, and determining whether the sample has pathogenic microorganisms or not and the DNA (Deoxyribonucleic Acid) concentration of the pathogenic microorganisms. By adopting the rapid pathogenic microorganism detection method disclosed by the invention, whether the sample has the pathogenic microorganisms or not and the content of the pathogenic microorganisms can be detected accurately, the detection analysis method is high in sensitivity, rapid in detection, simple in operation procedure and easy to implement, and rapid analysis on large samples can be achieved.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of cause of disease based on analysis PCR accessory substance pyrophosphoric acids
Method for rapid inspecting animalcule, it is adaptable to the specific pathogenic microorganisms such as food security, drinking water safety monitoring, clinical sample diagnosis
Rapid identification.
Background technology
Cause the invasive organism of human diseases and food poisoning (such as:Salmonella, staphylococcus, streptococcus, pair are molten
Courageous and upright vibrios, proteus, Shigella, avian influenza virus, Aspergillus flavus and virus, foot and mouth disease virus etc.) detection food
Product, health and medical field have great importance.Traditional cultivation detects complex operation step, time-consuming, it is impossible to meet
Quick detection is in the demand of diagnosis.
Molecular biology provides possibility for the quick detection of invasive organism.In principle, for certain microorganism
For qualitative analysis, the existing ripe molecular biology methods being widely used very and technology are roughly divided into two more
Class.One class is real-time tracking PCR (PCR)：Such as Real-time quantitative PCR, real-time melting curve analysis etc.；Separately
It is analyzed after the completion of one class is PCR, to PCR primer：As nucleic acid sequence analysis goldstandard Sanger DNA sequencing technologies,
DNA chip technology.Above-mentioned two class all refers to PCR and respectively has quality, and the former is quick but needs expensive instrument and reagent；The latter
It is cumbersome, time-consuming.Quick, the inexpensive detection of development specific nucleic acid sequence fragment is increasingly received in industries such as health, food
To pay attention to and it is required.
Round pcr is a kind of Protocols in Molecular Biology for amplifying the specific DNA fragmentation of amplification, can be by micro DNA
It is significantly increased.However, in existing molecular Biological Detection microbial technique, the object of all analyses is in PCR reactions
The DNA sequence dna of generation, its theoretical amount is the 2 of sample amplifying nucleic acid copy numberN-1(N is PCR cycle number), if using letters such as electrophoresis
Single, quick method analysis, then sample amplifying nucleic acid copy number need to reach hundreds of ability and clearly judged by electrophoretic band
Whether PCR reactions occur, and effective detection cannot be implemented to low abundance sample.
In fact, during PCR, often synthesizing nucleotides, a PCR primer will extend a bases longs, while
Also an accessory substance pyrophosphoric acid molecule can be supervened.If every a primer extends 100 bases during PCR,
A pair of DNA profilings will produce 200 pyrophosphoric acid molecules in a PCR process；That is during PCR, the pyrophosphoric acid of generation
Molecule amount is generally the hundred times of DNA.Therefore, if detection DNA is suitable with the sensitivity of pyrophosphoric acid, detection pyrophosphoric acid point
The sensitivity of son should improve hundred times than detection DNA molecular.Therefore, selection analysis PCR accessory substances pyrophosphoric acid, rather than PCR is produced
Its sensitivity of thing DNA fragmentation is bigger, is more suitable for the analysis of low abundance nucleic acid sequence.
Existing very many analysis methods are used for the quantitative analysis tech of pyrophosphoric acid, using the allusion quotation that pyrophosphoric acid is analyzed
Type example is sequenced for burnt.The technology is that the DNA fragmentation after PCR is expanded is detected, i.e., by obtaining the single-stranded of PCR primer
DNA, then sequencing by hybridization primer, sequencing primer extends nucleotides and is sequenced, and the acquisition that signal is sequenced is by nucleotides
What the pyrophosphoric acid molecule that extension is discharged was realized：By analyze in single sequencing reaction whether have pyrophosphoric acid produce and its
The amount of generation may determine that the number whether sequencing reaction has nucleotides to synthesize and synthesize.Its principle is that reaction substrate is 5'-
Phosphosulfate, fluorescein, in the presence of adenosine triphyosphate sulfurylase, pyrophosphoric acid can be with 5'- phosphosulfate knots
Conjunction forms adenosine triphyosphate, under the catalysis of luciferase, the adenosine triphyosphate of generation again can and fluorescence
Element combines to form oxyluciferin, while producing visible ray.By detection means can obtain one detection peak, the height of peak value and
The amount of pyrophosphoric acid is directly proportional.But the DNA fragmentation after typically PCR is expanded in the prior art is detected, i.e., by obtaining
The single stranded DNA of PCR primer, then sequencing by hybridization primer, sequencing primer extends nucleotides and is sequenced, and the acquisition of signal is sequenced
It is that the pyrophosphoric acid molecule discharged by nucleotides extension is realized, this detection still has operating process complexity, consumption
The problems such as duration and test limit.
The content of the invention
Goal of the invention：Have in microorganism detection that operating process is complicated, time-consuming and test limit for prior art
The problems such as, the present invention provides a kind of Detection Method of Pathogenic Microorganism based on analysis PCR accessory substance pyrophosphoric acids, the method root
According to the pathogenic microorganism of detection, one section of retrieval represents the nucleic acid sequence fragments of the microorganism feature and sets from common data base
A pair special PCR primers are counted, by expanding to sample genome specific PCR and the total pyrophosphoric acid produced during PCR is expanded is analyzed
Judge PCR reactions whether occur with occur number, so as to whether derive containing the specific microorganism and its DNA concentration.
The determination method sensitivity is high, and detection is fast, and operating process is simple, easily implements, and quick analysis can be implemented to large sample.
Technical scheme：To achieve these goals, it is a kind of based on analysis PCR accessory substance pyrophosphoric acids as described in the present invention
Detection Method of Pathogenic Microorganism, it is characterised in that comprise the following steps：
(1) according to the pathogenic microorganism of detection, one section of nucleic acids characteristic sequence for representing the microorganism is retrieved from database
Column-slice section；
(2) for this feature nucleic acid sequence fragments, a pair special PCR primers are designed；
(3) sample microbial genome is extracted, and enters performing PCR with special PCR primer and expanded；
(4) pyrophosphoric acid in analysis PCR amplifications；
(5) according to analysis result judge PCR reactions whether occur with occur number, determine whether micro- containing this in sample
Biological and its content.
Wherein, the nucleic acids characteristic sequence fragment described in step (1) is that the microorganism that needs are detected uniquely has, other are given birth to
The nucleic acid sequence information that do not have in thing and can be obtained by common data base.
The amplifications of PCR described in step (3) are special, nucleic acids characteristic sequence fragments in an amplification step (1), do not expand it
Its sequence fragment.
Pyrophosphoric acid is by pyrophosphoric acid chemiluminometry, Direct Analysis during PCR amplifications are analyzed described in step (4)
Total pyrophosphoric acid in PCR primer is realized.
Change of the pyrophosphoric acid chemiluminometry by being implemented using pyrophosphoric acid detection kit or by preparing
Luminescence reagent is learned to implement.
The pyrophosphoric acid detection kit is the pyrophosphoric acid detection kit of SIGMA-ALDRICH companies.
Further, the chemical illuminating reagent Main Ingredients and Appearance of the preparation is：0.01~0.5M Tris-acetate (pH
7.7), 10~50mM EDTA, 1~5mM Mg (Ac)2, 0.02%~0.1% bovine serum albumin(BSA) (BSA), the sulphur of 1~5mM bis- Soviet Union
Sugar alcohol (DTT), 10~50mM 5'- phosphosulfates (APS), 0.2~1.0mg/mL polyvinylpyrrolidones (PVP), 2~10mM
Fluorescein (D-luciferin), 50~800mM luciferases.Usually 0.01M Tris-acetate (pH 7.7), 20mM
EDTA,1mM Mg(Ac)2, 0.02% bovine serum albumin(BSA) (BSA), 1mM dithiothreitol (DTT)s (DTT), 10mM 5'- phosphosulfates
(APS), 0.4mg/mL polyvinylpyrrolidones (PVP), 4mM fluoresceins (D-luciferin), 800mM luciferases.
Further, in the pyrophosphoric acid chemiluminometry optical signal detection antigen photodiode or electricity
Lotus coupling element (CCD)；Can be with single detection, it is also possible to parallel detection.
Wherein, described in step (5) determine sample in whether containing the microorganism be by set pyrophosphoric acid CV values come
Judge：Pyrophosphoric acid analyte signal intensity is judged to have more than CV values, correspond to sample therewith in be, containing the microorganism, to be less than
CV values are judged to nothing, correspond to be free from the microorganism in sample therewith；
The quantitative analysis that the content of microorganisms in sample is determined described in step (5) refers to first pass through a series of microorganisms
Concentration known after setting up the relation curve of concentration and signal intensity, then analyzes the signal that sample is obtained by step (1)-(5)
Intensity, tries to achieve the specific concentration of the microorganism from relation curve.
The hundreds of times of pyrophosphoric acids of amount of DNA are produced during PCR-based of the present invention for judging whether final PCR reactions are sent out
The raw and extent of reaction, while whether containing certain microorganism and its content in the sample of participation PCR reactions of retrodicting out.Due to
Pyrophosphoric acid detection is ripe technology, and in the market has corresponding kit to sell (SIGMA-ALDRICH companies etc.).Therefore, only
The quantitative analysis that a simple optical signal detecting device can just be realized to pyrophosphoric acid need to be built.
Beneficial effect：Prior art is compared, and the present invention is a large amount of pyrophosphoric acids for producing during direct detection PCR to be sentenced
Whether disconnected PCR reactions occur；It is that the DNA fragmentation after PCR is expanded is carried out and technology discloses pyrosequencing techniques now
Detection, i.e., by obtaining the single stranded DNA of PCR primer, then sequencing by hybridization primer, sequencing primer extends nucleotides and is sequenced,
And the acquisition that signal is sequenced is the pyrophosphoric acid molecule realization discharged by nucleotides extension.Therefore, although be all inspection
Pyrophosphoric acid molecule is surveyed, difference is：1st, disclosed pyrosequencing techniques are the single sequencing reactions of real-time detection in the prior art
Pyrophosphoric acid molecule；And present invention detection is that PCR reacts the total pyrophosphoric acid molecule for producing.2nd, the object of detection is different, existing
Disclosed in technology pyrosequencing techniques detection pair as if PCR primer DNA sequence dna；And the present invention detection pair as if PCR
The pyrophosphoric acid of product.
The present invention has following advantages：
1st, this feature nucleic acid sequence fragments of present invention selection microorganism design specific primer, by detecting PCR by-products
Whether pyrophosphoric acid in thing, can accurately detect in sample contain the microorganism and its DNA concentration.
2nd, the present invention tracks PCR and reacts using the pyrophosphoric acid of detection hundreds times DNA molecular, its remolding sensitivity traditional detection
The sensitivity of DNA molecular method is high.
3rd, using pyrophosphoric acid content in direct detection PCR primer, PCR is completed the present invention, and sample can be completed in 3 minutes
Analysis, its detection speed is faster than traditional detection DNA molecular method.
4th, the method for the present invention can implement quick analysis to large sample, can be used for primary dcreening operation, can be used for detection.
5th, the present invention can realize the quantitative analysis of pyrophosphoric acid using easy optical detection apparatus, and operating process is simple,
Easily implement.
Brief description of the drawings
Fig. 1 is the standard of shigella dysenteriae DNA profiling concentration C (ng/ μ L) and fluorescence signal intensity (Y) in the embodiment of the present invention 1
Curve；
Fig. 2 is the PCR primer agarose gel electrophoresis testing result of different content shigella dysenteriae in the embodiment of the present invention 1.
Specific embodiment
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment 1：The detection of shigella dysenteriae in chicken
1st, genomic DNA sample preparation：The bacterium on chicken surface is rinsed using PBS (PH=7.2), is then used
The DNA of bacteria that DNA of bacteria extracts kit will be rinsed is extracted, and Cord blood is standby.
2nd, PCR primer design and PCR reactions
For shigella dysenteriae nucleic acids characteristic sequence fragment, the ipaH genes of shigella dysenteriae are the genes of coding secretion virulence protein,
It is closely related with the bacteria pathogenic, it is that Shigella is distinctive, according to GenBank accession no.M76445.1 designs just
To primer and reverse primer：
SEQ ID NO 1 are forward primer F:5 ' ACTATTGGCTCTGGATGTT 3 ',
SEQ ID NO 2 are reverse primer R:5’GGGCGATGGAGTGTATT 3’；
(sequence of amplification gene is retrieved in GeneBank databases, is submitted in primer-design software Primer5.0
Row design of primers, its specificity can be by NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) carry out
Homologous sequence is compared), it is desirable to primer specificity is strong, without or less primer dimer, misquote.
The primer of design is synthesized and following PCR reactions are done：
(1) measure of standard curve：The PCR amplification system of 25 μ L is included：Genomic DNA is respectively 126,1.26,1.26
×10-2、1.26×10-4、1.26×10-6, 0ng/ μ L, 0.2mM dNTP, 0.8 μM of forward primer and 0.8 μM of reverse primer, 2U
Taq archaeal dna polymerases, 1 × amplification buffer, 1.5mM MgCl2.Amplification condition：94 DEG C of predegenerations 5 minutes, 35 thermal cycles
(94 DEG C of 30 seconds~54 DEG C of denaturation, 30 seconds~72 DEG C of annealing extend 30 seconds), last 72 DEG C extend 5 minutes.
(2) detection of actual sample：The PCR amplification system of 25 μ L is included：200ng genomic DNAs, 0.2mM dNTP, 0.8
μM forward primer and 0.8 μM of reverse primer, 2U Taq archaeal dna polymerases, 1 × amplification buffer, 1.5mM MgCl2.Amplification bar
Part：94 DEG C of predegenerations 5 minutes, 35 thermal cycles (94 DEG C of 30 seconds~54 DEG C of denaturation, 30 seconds~72 DEG C of annealing extend 30 seconds), finally
72 DEG C extend 5 minutes.
3rd, PCR primer detection
(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, 0.02% bovine serum albumin(BSA) (BSA), 1mM dithiothreitol (DTT)s (DTT), 10mM 5'- phosphosulfates
(APS), 0.4mg/mL polyvinylpyrrolidones (PVP), 4mM fluoresceins (D-luciferin), 800mM luciferases.
(2) PCR primer detection
A. Weak light investigating instrument is opened, temperature and high-voltage value is set.
B. prepare 1.5mL centrifuge tubes, add 400 μ L chemical luminous system (S-mix) solution.
C. check 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 lid, opens shutter, measures the background signal of chemical luminous system solution.
D. close fast opening behind the door to cover, take out centrifuge tube.The PCR of 1 μ L is added in chemical luminous system solution is added
Amplified production, vibration is well mixed.Sample room is then placed in, shutter is opened, measurement obtains the fluorescence signal of pcr amplification product
Value.The data that record measurement is obtained.
E. after measurement is finished, shutter, measuring instrument are closed.
F. CV values according to obtained by measurement under shigella dysenteriae DNA normal concentrations, by the fluorescence signal value of institute's test sample product and CV values
Evolution is compared, signal intensity is judged to have more than CV values, correspond to sample therewith in be, containing the microorganism, to judge less than CV values
It is nothing, corresponds to be free from microorganism in sample therewith.
(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 concentrations
Signal value, the signal intensity for measuring sample is positive more than 45000, to represent and contain the microorganism in the sample.Signal
It is slight pollution that intensity is located between 45000 and 56000；It is moderate dirt that signal intensity is located between 56000 and 88000
Dye, signal intensity is serious pollution more than 88000.
The fluorescence signal intensity of PCR primer under the different DNA profiling concentration of table 1.
B. the drafting of standard curve：The fluorescence signal intensity (Y) measured according to known dna template concentrations (C) in table 1 is drawn
Experiment curv Fig. 1, the fitting equation that fitting is obtained is Y=11750X+86300, and X is the logarithm (lgC) of DNA concentration, and Y is survey
The fluorescence signal intensity for obtaining subtracts the fluorescence signal intensity (24000) of blank.
C. actual sample testing result：The signal intensity that the present embodiment 2 measures sample 1 is 45000, according to standard curve
(Fig. 1), it is known that the shigella dysenteriae DNA concentration of its detection is 3.98 × 10-6Ng/ μ L, are slight pollution；The signal intensity of sample 2 is
22000, shigella dysenteriae is not detected, without pollution with blank 24000 quite.
Comparative example 1：Shigella dysenteriae contrasting detection method
(1) the PCR primer electrophoresis detection of shigella dysenteriae genomic DNA known to
1st, genomic DNA sample preparation：The bacterium on chicken surface is rinsed using PBS (PH=7.2), is then used
The DNA of bacteria that DNA of bacteria extracts kit will be rinsed is extracted and determines the concentration of genomic DNA.
2nd, PCR primer design and PCR reactions
For shigella dysenteriae nucleic acids characteristic sequence fragment, the ipaH genes of shigella dysenteriae are the genes of coding secretion virulence protein,
It is closely related with the bacteria pathogenic, it is that Shigella is distinctive, according to GenBank accession no.M76445.1 designs just
To primer and reverse primer：
SEQ ID NO 1 are forward primer F:5 ' ACTATTGGCTCTGGATGTT 3 ',
SEQ ID NO 2 are forward primer R:5’GGGCGATGGAGTGTATT 3’；
(sequence of amplification gene is retrieved in GeneBank databases, is submitted in primer-design software Primer5.0
Row design of primers, its specificity can be by NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) carry out
Homologous sequence is compared), it is desirable to primer specificity is strong, without or less primer dimer, misquote.
The primer of design is synthesized and following PCR reactions (being not added with DNA profiling for blank) are done：
The PCR amplification system of 25 μ L is included：Genomic DNA template concentration is respectively 126,1.26,1.26 × 10-2、1.26
×10-4、1.26×10-6, 0ng/ μ L, 0.2mM dNTP, 0.8 μM of forward primer and 0.8 μM of reverse primer, 2U Taq DNA gather
Synthase, 1 × amplification buffer, 1.5mM MgCl2.Amplification condition：94 DEG C of predegenerations 5 minutes, 35 thermal cycles (94 DEG C of denaturation 30
Second~54 DEG C of 30 seconds~72 DEG C of annealing extensions 30 seconds), last 72 DEG C extend 5 minutes.
3rd, PCR primer agarose gel electrophoresis detection
5 microlitres of PCR primers are splined on the Ago-Gel that concentration is 1%, row agarose gel electrophoresis are entered to PCR primer
(deposition condition：120V, 20 minutes), whether observation band is clear, testing result such as Fig. 2.In Fig. 2, different content shigella dysenteriae
PCR primer agarose gel electrophoresis testing result.M, 1,2,3,4,5,6 are respectively the DNA fragmentation of known length in figure
(Marker), pcr template concentration is respectively 126,1.26,1.26 × 10-2、1.26×10-4、1.26×10-6、0ng/μL。
(2) shigella dysenteriae culture detection method
1st, BPW culture mediums are prepared：Weigh BPW culture medium 20.1g, add distilled water 1L, agitating heating is boiled to completely molten
Solution, each conical flask is dispensed into 90mL/ bottles, and the 15min that sterilized in 121 DEG C, is cooled to normal temperature.
2nd, after culture medium is cooled to room temperature, ultraviolet sterilization is carried out 15 minutes to culture medium and whole room, after the 60min that turns off the light
Use.
3rd, bacterium is increased in advance：Chicken meat sample 10g to the conical flask containing BPW culture mediums is weighed, and is closed the lid in time, under 150rpm
Vibration 10min, in 36 DEG C of ± 1 DEG C of culture 18h or so.
4th, TTB enrichment liquids are prepared：Take TTB enrichment liquids basis 93.6g, add distilled water 1L, agitating heating is boiled to complete
Dissolving, 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 reagents iodine solution and brilliant green each one (preceding 75% alcohol swab sterilization cillin bottle of unlatching are added in 100mL basal mediums
Surface), it is well mixed.
5th, bacterium is increased
6th, it is inoculated with and cultivates：The sample mixture cultivated is shaken gently for, 1mL is pipetted, transferred species is in 9mL TTB enrichment liquids
It is interior, in 42 DEG C of ± 1 DEG C of culture 18h~24h.
7th, observation culture phenomenon.
8th, shigella dysenteriae chromogenic culture medium flat board is prepared.
9th, dry powder 4.75g in bottle is taken, water dissolves is distilled with 100mL, can be scaled up or reduce.
10th, 100 DEG C are mixed and heated to, are heated with stirring to and are completely dissolved, be cooled to 50 DEG C and be down flat plate.
11st, sample setting-out or rubbing method are inoculated with, and 37 DEG C are cultivated 24 hours.
12nd, colony colour is observed.
It is of the invention based on analysis PCR by-products by the electrophoresis detection comparative analysis with known shigella dysenteriae DNA concentration sample
The genomic DNA template least concentration that the Detection Method of Pathogenic Microorganism of thing pyrophosphoric acid can be detected is 1.26 × 10- 6Ng/ μ L, and traditional PCR primer electrophoresis assays cannot be 1.26 × 10 to genomic DNA template concentration-2The sample of ng/ μ L
Detected, thus it is sensitiveer.
By the comparative analysis of actual sample culture detection method, the disease based on analysis PCR accessory substance pyrophosphoric acids of the invention
Pathogenic microorganism method for quick is consistent with culture detection method result.
Culture detection method is a kind of strict, 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；The electrophoresis assays of microorganism PCR primer are a kind of qualitative points of current quick detection
Analysis method, relative to the electrophoresis assays of microorganism PCR primer, the method for embodiment 1 not only take it is shorter, and can be to detecting
Microorganism carries out quantitative analysis.Therefore, Detection Method of Pathogenic Microorganism of the present invention based on analysis PCR accessory substance pyrophosphoric acids
Detection is fast, and operating process is simple, easily implements, and quick analysis can be implemented to large sample, is especially suitable for the industries such as food, health
Microbial rapid detection.
Embodiment 2
Embodiment 2 is used and the identical method of embodiment 1, and difference is that pyrophosphoric acid chemiluminometry is used
The pyrophosphoric acid detection kit of SIGMA-ALDRICH companies purchase, testing result is similar to Example 1.
SEQUENCE LISTING
<110>Southeast China University
<120>Detection Method of Pathogenic Microorganism based on analysis PCR accessory substance pyrophosphoric acids
<130> 2017.01.20
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213>It is artificial synthesized
<400> 1
actattggct ctggatgtt 19
<210> 2
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 2
gggcgatgga gtgtatt 17
Claims (10)
1. it is a kind of based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is characterised in that including as follows
Step：
(1) according to the pathogenic microorganism of detection, one section of nucleic acids characteristic tract for representing the microorganism is retrieved from database
Section；
(2) for this feature nucleic acid sequence fragments, a pair special PCR primers are designed；
(3) sample microbial genome is extracted, and enters performing PCR with special PCR primer and expanded；
(4) pyrophosphoric acid in analysis PCR amplifications；
(5) according to analysis result judge PCR reactions whether occur with occur number, determine whether contain the microorganism in sample
And its DNA concentration.
2. according to claim 1 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, the nucleic acids characteristic sequence fragment described in step (1) be need detection microorganism uniquely have, in other biologies not
With and by the nucleic acid sequence information that can obtain of common data base.
3. according to claim 1 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, the amplifications of PCR described in step (3) are special, nucleic acids characteristic sequence fragments in an amplification step (1), do not expand it
Its sequence fragment.
4. according to claim 1 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, pyrophosphoric acid is by pyrophosphoric acid chemiluminometry, Direct Analysis PCR during PCR amplifications are analyzed described in step (4)
Total pyrophosphoric acid in product is realized.
5. according to claim 4 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, chemistry of the pyrophosphoric acid chemiluminometry by being implemented using pyrophosphoric acid detection kit or by preparing
Luminescence reagent is implemented.
6. according to claim 5 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, the pyrophosphoric acid detection kit is the pyrophosphoric acid detection kit of SIGMA-ALDRICH companies.
7. according to claim 5 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, the chemical illuminating reagent Main Ingredients and Appearance of the preparation is：0.01~0.5M Tris-acetate (pH 7.7), 10~
50mM EDTA, 1~5mM Mg (Ac)2, 0.02%~0.1% bovine serum albumin(BSA) (BSA), 1~5mM dithiothreitol (DTT)s
(DTT), 10~50mM 5'- phosphosulfates (APS), 0.2~1.0mg/mL polyvinylpyrrolidones (PVP), 2~10mM fluorescence
Plain (D-luciferin), 50~800mM luciferases.
8. according to claim 4 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, it is special
Levy and be, the detection antigen photodiode or charge coupled cell of optical signal in the pyrophosphoric acid chemiluminometry
(CCD)；Can be with single detection, it is also possible to parallel detection.
9. according to claim 1 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, step
(5) whether containing the microorganism judged by the pyrophosphoric acid CV values for setting in determination sample described in：Pyrophosphoric acid is analyzed
Signal intensity is judged to have more than CV values, correspond to sample therewith in be containing the microorganism, less than CV values be judged to without, therewith
Correspond to be free from the microorganism in sample.
10. according to claim 1 based on the Detection Method of Pathogenic Microorganism for analyzing PCR accessory substance pyrophosphoric acids, its
It is characterised by, the quantitative analysis that the content of microorganisms in sample is determined described in step (5) refers to first pass through a series of micro- lifes
Thing concentration known after setting up the relation curve of concentration and signal intensity, then analyzes the letter that sample is obtained by step (1)-(5)
Number intensity, tries to achieve the specific concentration of the microbial DNA from relation curve.
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| CN109797195A (en) * | 2019-01-18 | 2019-05-24 | 东南大学 | Freeze-draw method rapid detection method and its application based on analysis PCR by-product pyrophosphoric acid |
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