CN107419012A - Detect pseudomonas aeruginosa and lasB primer, kit and method in water - Google Patents
Detect pseudomonas aeruginosa and lasB primer, kit and method in water Download PDFInfo
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Abstract
The invention discloses pseudomonas aeruginosa and lasB primer, kit and method in detection water, the sequence and probe sequence of primer are respectively：Sense primer P.AF, anti-sense primer P.AR, probe P.AP, sense primer lasBF, anti-sense primer lasBR, probe lasBP.The invention aims to overcome weak point of the prior art, there is provided a kind of primer and component and reasonable mixture ratio, easy to use, detection is quick, accurately, the kit of pseudomonas aeruginosa and lasB genes water is detected suitable for dual ddPCR；Another object of the present invention is to provide a kind of method of pseudomonas aeruginosa and LasB genes in detection water using mentioned reagent box, and this method is easy to operate, quick, and testing result is accurate.
Description
Technical field
The present invention relates to one kind to detect pseudomonas aeruginosa and its important virulence factor elastase LasB genes in water
Dual droplet type digital pcr detection kit, it is false using verdigris in mentioned reagent box detection drinking water the invention further relates to one kind
The method of monad and lasB.
Background technology
Pseudomonas aeruginosa Pseudomonas aeruginosa are former to claim Pseudomonas aeruginosa.It is extensive in distributed in nature, for soil
One of most common bacterium present in earth.Various water, air, the skin of normal person, respiratory tract and enteron aisle etc. have this bacterium to deposit
.Essential condition existing for this bacterium is moist environment.The bacterium is a kind of common environmental microorganism, because to nutritional requirement not
Height, excel at leveraging various carbon sources and ammonification compound as nitrogen source, so wide in the environment such as water, soil, food and hospital
General presence.The limit of the U.S., Canada, Europe, Japan, Brazil and the World Health Organization to the green pseudomonad content of copper in drinking water
Determine MPN<3/L must not detect per 250mL.China《National food safety standard packs drinking water》(GB19298-2014), then
Provide in 5 water samples, 250mL must not detect pseudomonas aeruginosa in each sample.
In October, 2009,《Natural mineral water》Etc. aqueous phase close national food safety standard successively all by bacterium
Fall this total index to delete.Although this reform has practical significance, objectively so that water manufacturing enterprise simplifies
Disinfectant program, substantially increase the exceeded probability of pseudomonas aeruginosa.In recent years, various regions are even more that frequency shows the green false list of copper in drinking water
The exceeded situation of born of the same parents.
There are some researches show the lasB genes of, pseudomonas aeruginosa may play unusual effect, elastoser LasB,
By lasB gene codes, total length 1497bp, 498aa being encoded, preceding 23aa is signal peptide, and 24~498aa is proelastase,
Final product is 301aa, is secreted by II type excretory system to extracellular.Its biosynthesis is by multiple genes such as lasA and QS systems
Regulation and control etc..Elastoser is the important virulence factor of pseudomonas aeruginosa, and can degrade elastin laminin, causes the damage of pulmonary parenchyma
Wound and bleeding, it is relevant with charrin disease diffusion；Can be degraded complement and suppression endogenous antibacterial material activity.
, quick detection accurate to pseudomonas aeruginosa that the strict control of the drinking water quality of production needs, at the same it is false to verdigris
Monad is propagated risk and assessed.Special primer, probe of the invention by designing pin pseudomonas aeruginosa and lasB genes
Combination, it may be appreciated that in water in the distribution of pseudomonas aeruginosa and bacterial strain lasB genes popularity.
At present, the related many food security standards of drinking water require carries out quantitative detection to pseudomonas aeruginosa,
But the quantitative detection for pseudomonas aeruginosa mainly or after being cultivated by conventional method counts, but conventional method is present
The shortcomings of process is cumbersome, and the cycle is long.It is not only cumbersome and conventional PCR method needs PCR to carry out electrophoresis after expanding, and not
Quantitative detection can be realized.At present, Southern blot and real-time fluorescence quantitative PCR are two kinds of conventional copy number of foreign gene
Analytical technology, it is widely used in copy number of foreign gene analysis.But there is also certain defect for both approaches.For example,
Workload is big, the cycle is long when Southern blot methods are analyzed, operation requires that height, accuracy are poor, especially for multicopy
The analysis of gene, it is as a result easily less than normal.Quantitative fluorescent PCR (qRT-PCR) is necessarily dependent upon when analyzing copy number of foreign gene
It is pure that the gene of standard curve and known copy number, simply a kind of relative quantitation method, and the quality of standard curve are vulnerable to DNA
The factors such as degree, the concentration of primer and probe, the response inhabitation factor influence；In addition, standard curve must be based on standard substance
Establish, and the limitednumber of standard substance and expensive price are not applied for all research.
Droplet digital pcr is a kind of new absolute quantitation technology of rising in recent years, and it is entered based on single-molecule PCR method
The nucleic acid quantification that row counts, is a kind of method of absolute quantitation.The main miniflow using the popular research field of present analysis chemistry
Control or droplet method, the nucleic acid solution after Macrodilution are dispersed in the microreactor or droplet of chip, each reactor
Nucleic acid-templated number be less than or equal to 1.So pass through after PCR cycle, have the reactor of a nucleic acid templates just
Fluorescence signal can be provided, does not just have fluorescence signal without the reactor of template.According to relative scale and the volume of reactor, so that it may
To extrapolate the gene copy number of original solution.
But the analysis work report of the copy number based on droplet digital pcr platform is less, for pseudomonas aeruginosa and
The detection of lasB genes, which just more has no, to be reported.The present invention be based on droplet type ddPCR platforms, establish pseudomonas aeruginosa and
Its lasB gene copy number analysis method, result of study provide new method to analyze the quantitative detection of drinking water risks and assumptions
And reference, also provide a technical support means for the control of drinking water routine quality.
The content of the invention
The invention aims to overcome weak point of the prior art, there is provided a kind of primer and component and proportioning are closed
Reason, easy to use, detection is quick, accurately, the examination of pseudomonas aeruginosa and lasB genes water is detected suitable for dual ddPCR
Agent box；
Another object of the present invention is to provide pseudomonas aeruginosa and LasB bases in a kind of detection water using mentioned reagent box
The method of cause, this method is easy to operate, quick, and testing result is accurate.
In order to achieve the above object, the present invention uses following scheme：
Pseudomonas aeruginosa and lasB gene primers in a kind of water, it is characterised in that the sequence and probe sequence of the primer point
It is not：
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer lasBF：AGCCATCACCGAAGTCAAGG
Anti-sense primer lasBR：CGTTCAGTGGCGAATAAGCG
Probe lasBP：CGACAGCAAGACCACGCCGT.
The kit of pseudomonas aeruginosa and lasB genes in a kind of ddPCR detections water, it is characterised in that in the kit
Reaction system includes following components：The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and lasB is forward and reverse draws
Each 0.1-1.0 μ L of thing, each 0.1-1.0 μ L of probe, the μ L of DNA profiling 4.0.
A kind of ddPCR detection pseudomonas aeruginosas and the method for LasB genes, it is characterised in that comprise the following steps：
A, sample DNA is extracted；
B, each reactive component is added into above-mentioned reaction system, then adds 70.0 μ l mineral oil, droplet is transferred to after mixing
Droplet is automatically generated on generator；
C, caused droplet is carefully transferred completely into 96 hole reaction plate PCR reaction tubes；Again by 96 hole reaction plates in sealer
Instrument upper sealing film, it is placed in regular-PCR instrument and enters performing PCR reaction.
D, droplet fluorescence detector application software is opened, 96 hole reaction plates after PCR reactions are terminated, which are inserted directly into, to be set
It is standby, the PCR response situations per droplet in PCR reaction tubes are detected, the copy of testing gene is finally calculated according to amber pine distribution law
Number.
The method of detection pseudomonas aeruginosa and lasB genes as described above, it is characterised in that PCR is expanded anti-in step B
Program is answered to carry out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
The method of detection pseudomonas aeruginosa and lasB genes as described above, it is characterised in that extract sample described in step A
Product DNA's comprises the following steps that：
50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
In 50mL centrifuge tube, with 10000rpm is centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；5mL CTAB are added to carry
Liquid is taken, is fully mixed.65 DEG C of incubation 30min, vibrate frequently；8000rpm room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter
In 2ml centrifuge tubes；Add after 700 μ L chloroforms and use forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 are to newly
In 2ml centrifuge tubes；Add 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed
Even, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature is placed
20min, 13000rpm centrifuge 10min, supernatant discarding, add the ethanol solutions of 500 μ L 70% washing precipitation, are dissolved in 50 μ L
In TE solution.
Sensitivity and Specificity is tested in the present invention：
Sequence verification is carried out to positive amplification product using the methods of sequencing, as a result positive amplification Product Sequence is carried out
When Blast compares, sequence with Genbank aim sequence very high homologies.The reference strain genomic DNA of 10 times of dilutions is added
Enter previous reaction system, repeat that there is repeatability very well in experiment display detection sample.Dilute template concentrations logarithm value and
Also it is in good linear relationship R between copy numerical value2≥0.95.Illustrate that this method has preferable accuracy and good stabilization
Property.
The present invention compared with prior art, has advantages below:
1) reagent constituents and reasonable mixture ratio of the present invention, easy to use, detection is quick, accurately, is quantitatively examined suitable for ddPCR
Survey pseudomonas aeruginosa and lasB genes；
2) detection method simplifies testing process, and need not make standard curve, substantially reduces detection week
Phase, detection time shorten two days or so than tradition culture method of counting；
3) detection method whole process is without using standard curve, and direct with new-generation sequencing slitless connection,
Absolute quantification analysis can perform to gene copy number.
4) digital pcr detecting system is handled by droplet, can greatly reduce the interference of background and matrix, and sensitivity can
With as little as 1 copy, therefore, the slight change to low concentration mrna concentration carries out detection accurate and that repeatability is good.
5) pseudomonas aeruginosa and lasB genes simultaneously can be achieved in same reaction system using detection method
Precisely detected, it is easy to operate, quick.With preferable industrialization prospect.
Embodiment
The present invention is described further with reference to embodiment：
Embodiment 1
The primer of pseudomonas aeruginosa and lasB genes in present invention detection water, sequence are respectively：
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer lasBF：AGCCATCACCGAAGTCAAGG
Anti-sense primer lasBR：CGTTCAGTGGCGAATAAGCG
Probe lasBP：CGACAGCAAGACCACGCCGT
Embodiment 2
The kit of pseudomonas aeruginosa and lasB genes in a kind of detection water of the present invention, wherein 20 μ L reaction system bags
Include following components：
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 1.0 μ L of the forward and reverse primers of LasB, visit
Each 1.0 μ L of pin, the μ L of DNA profiling 4.0.
Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer lasBF：AGCCATCACCGAAGTCAAGG
Anti-sense primer lasBR：CGTTCAGTGGCGAATAAGCG
Probe lasBP：CGACAGCAAGACCACGCCGT.
Embodiment 3
The kit of pseudomonas aeruginosa and lasB genes in a kind of detection water of the present invention, reaction system are included with the following group
Point：
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 0.1 μ L of the forward and reverse primers of LasB, visit
Each 0.1 μ L of pin, the μ L of DNA profiling 4.0.
Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer lasBF：AGCCATCACCGAAGTCAAGG
Anti-sense primer lasBR：CGTTCAGTGGCGAATAAGCG
Probe lasBP：CGACAGCAAGACCACGCCGT.
Embodiment 4
Pseudomonas aeruginosa and lasB genetic methods in present invention detection water, comprise the following steps：
A, sample DNA is extracted；
B, the 20.0 μ l reaction systems for adding each reactive component in embodiment 2, then add 70.0 μ l mineral oil, mix
After be transferred on drop generator and automatically generate droplet；
C, caused droplet is carefully transferred completely into 96 hole reaction plate PCR reaction tubes；96 hole reaction plates are being sealed again
Film instrument upper sealing film, it is placed in regular-PCR instrument and enters performing PCR reaction.
D, droplet fluorescence detector application software is opened, 96 hole reaction plates after PCR reactions are terminated, which are inserted directly into, to be set
It is standby, the PCR response situations per droplet in PCR reaction tubes are detected, the copy of testing gene is finally calculated according to amber pine distribution law
Number.
Described fluorescent PCR amplification is carried out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Embodiment 5
Pseudomonas aeruginosa and lasB genetic methods in present invention detection water, comprise the following steps：
50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
In 50mL centrifuge tube, with 10000rpm is centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；5mL CTAB are added to carry
Liquid is taken, is fully mixed.65 DEG C of incubation 30min, vibrate frequently；8000rpm room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter
In 2ml centrifuge tubes；Add after 700 μ L chloroforms and use forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 are to newly
In 2ml centrifuge tubes；Add 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed
Even, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature is placed
20min, 13000rpm centrifuge 10min, supernatant discarding, add the ethanol solutions of 500 μ L 70% washing precipitation, are dissolved in 50 μ L
In TE solution.
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 1.0 μ L of the forward and reverse primers of lasB, visit
Each 1.0 μ L of pin, the μ L of DNA profiling 4.0.Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer lasBF：AGCCATCACCGAAGTCAAGG
Anti-sense primer lasBR：CGTTCAGTGGCGAATAAGCG
Probe lasBP：CGACAGCAAGACCACGCCGT.
Enter performing PCR amplification, carry out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Embodiment 6
Pseudomonas aeruginosa and lasB genetic methods in present invention detection water, comprise the following steps：
50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to grip filter membrane with tweezers by membrane filtration
In 50mL centrifuge tube, with 10000rpm is centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；5mL CTAB are added to carry
Liquid is taken, is fully mixed.65 DEG C of incubation 30min, vibrate frequently；8000rpm room temperatures centrifugation 10min afterwards, takes 1ml supernatants to enter
In 2ml centrifuge tubes；Add after 700 μ L chloroforms and use forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 are to newly
In 2ml centrifuge tubes；Add 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed
Even, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature is placed
20min, 13000rpm centrifuge 10min, supernatant discarding, add the ethanol solutions of 500 μ L 70% washing precipitation, are dissolved in 50 μ L
In TE solution.
The μ L of wherein 2 × ddPCR Super Mix 10.0, pseudomonas aeruginosa and each 0.1 μ L of the forward and reverse primers of lasB, visit
Each 0.1 μ L of pin, the μ L of DNA profiling 4.0.Wherein primer sequence is as follows:
Sense primer P.AF:TGGTAGTCCACGCCGTAAA
Anti-sense primer P.AR:CAGACTGCGATCCGGACTACG
Probe P.AP:TCGACCGCCTGGGGAGTACG
Sense primer lasBF：AGCCATCACCGAAGTCAAGG
Anti-sense primer lasBR：CGTTCAGTGGCGAATAAGCG
Probe lasBP：CGACAGCAAGACCACGCCGT.
Enter performing PCR amplification, carry out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 60 DEG C of annealing 1min, carry out 40 circulations altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Interpretation of result
In development process of the present invention, while using the classical culture protocols of culture medium, carry out pseudomonas aeruginosa quantity survey
Fixed control.Test in triplicate, pseudomonas aeruginosa results averaged.As a result show, the present invention uses digital pcr method
To the copy number of pseudomonas aeruginosa in water, the total plate count result coefficient R detected with classical culture protocols2≥
99%.In addition, digital pcr of the present invention is enjoyed oneself to the full, single sample whole process detection time of method is 4 hours, and considerably shorter than existing traditional puts down
Plate culture method of counting, and detect pseudomonas aeruginosa simultaneously, moreover it is possible to quantitative detection is carried out to its lasB gene, can accurately be commented
Valency pathogenic bacteria and pathogenic bacteria carry the propagation risk of drug resistant gene, thus with good technical advantage and industrialized development before
Scape.
The general principle and principal character and advantages of the present invention of the present invention has been shown and described above.The skill of the industry
For art personnel it should be appreciated that the present invention is not limited to the above embodiments, described in above-described embodiment and specification is explanation
The principle of the present invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these
Changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent thereof.
Annex 1
Pseudomonas aeruginosa 16sRNA amplified production sequences
tggtagtc cacgccgtaaacgatgtcgactagccgttgggatccttgagatcttagtg
gcgcacgtaacgcgataagtcgaccgcctggggagtacggccgcaaggtt
aaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggt
ttaattcgaagcaacgcgaagaaccttacctggccttgacatgctgagaa
ctttccagagatggattggtgccttcgggaacagagacacaggtgctgca
tggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgtaacga
gcgcaacccttgtccttagttaccagcacctcgggtgggcactctaagga
gactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcat
ggcccttacggccagggctacacacgtgctacaatggtcggtacaaaggg
ttgccaagccgcgagtgggagctaatcccataaaaccgatcgtagtccgg atcgcagtctg
Virulence gene lasB gene amplification product sequences
agccatcaccgaagtcaagggtcccggcaagagcgtggcggcgcggcgcagcggccatttcgtcgccaacatcgccg
ccgacctgccgggcagcaccaccgcggcggtatccgccgagcaggtgctggcccaggccaagagcctgaaggcccag
ggccgcaagaccgagaatgacaaagtggaactggtgatccgcctgggcgagaacaacatcgcccaactggtctacaa
cgtctcctacctgattcccggcgagggactgtcgcggccgcatttcgtcatcgacgccaagaccggtgaagtgctcg
atcagtgggaaggcctggcccacgccgaggcgggcggccccggtggcaaccagaagatcggcaagtacacctacggt
agcgactacggtccgctgatcgtcaacgaccgctgcgagatggacgacggcaacgtcatcaccgtcgacatgaacgg
cagcaccaacgacagcaagaccacgccgttccgcttcgcctgcccgaccaacacctacaagcaggtcaacggcgctt
attcgccactgaacg。
SEQUENCE LISTING
<110>Cai is first complete
<120>Detect pseudomonas aeruginosa and lasB primer, kit and method in water
<130>Detect pseudomonas aeruginosa and lasB primer, kit and method in water
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Pseudomonas aeruginosa
<400> 1
tggtagtcca cgccgtaaa 19
<210> 2
<211> 21
<212> DNA
<213> Pseudomonas aeruginosa
<400> 2
cagactgcga tccggactac g 21
<210> 3
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 3
tcgaccgcct ggggagtacg 20
<210> 4
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 4
agccatcacc gaagtcaagg 20
<210> 5
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 5
cgttcagtgg cgaataagcg 20
<210> 6
<211> 20
<212> DNA
<213> Pseudomonas aeruginosa
<400> 6
cgacagcaag accacgccgt 20
Claims (5)
- A kind of 1. pseudomonas aeruginosa and gene lasB primer in detection water, it is characterised in that the sequence of primer and probe combination Row are respectively：Sense primer P.AF:TGGTAGTCCACGCCGTAAAAnti-sense primer P.AR:CAGACTGCGATCCGGACTACGProbe P.AP:TCGACCGCCTGGGGAGTACGSense primer lasBF：AGCCATCACCGAAGTCAAGGAnti-sense primer lasBR：CGTTCAGTGGCGAATAAGCGProbe lasBP：CGACAGCAAGACCACGCCGT.
- A kind of 2. pseudomonas aeruginosa and gene lasB digital pcr detection kit in detection water, it is characterised in that the reagent Reaction system includes following components in box：Wherein 2 × ddPCR Super Mix 10.0 μ L, pseudomonas aeruginosa and lasB is positive and negative To each 0.1-1.0 μ L of primer, each 0.1-1.0 μ L of probe, the μ L of DNA profiling 4.0.
- A kind of 3. pseudomonas aeruginosa and gene lasB method in detection water, it is characterised in that comprise the following steps：A, detect in water and extract sample DNA；B, each reactive component is added into reaction system as claimed in claim 2, then adds 70.0 μ l mineral oil, shifted after mixing Droplet is automatically generated on to drop generator；Take positive quality control and negative Quality Control in the kit respectively simultaneously, according to The processing of step A identicals method, obtains corresponding DNA profiling；C, digital pcr mixed liquor will be prepared and be made as the micro- reactions of Water-In-Oil PCR, and be transferred completely into 96 hole reaction plate PCR reactions Guan Zhong；Again by 96 hole reaction plates in sealer instrument upper sealing film, it is placed in regular-PCR instrument and enters performing PCR reaction；D, droplet fluorescence detector application software is opened, 96 hole reaction plates after PCR reactions are terminated are inserted directly into equipment, detect The PCR response situations of droplet in per PCR reaction tubes, the copy number of testing gene is finally calculated according to amber pine distribution law.
- 4. pseudomonas aeruginosa and gene lasB method in a kind of detection water according to claim 1, it is characterised in that The response procedures that PCR is expanded in step C are carried out according to the following steps：(1) 94 DEG C of pre-degeneration 3min；(2) 94 DEG C of denaturation 10s, 59 DEG C of annealing 1min, carry out 40 circulations altogether；(3) 98 DEG C of enzyme heat inactivation 10min；(4) 4 DEG C stop reaction.
- 5. a kind of pseudomonas aeruginosa and gene lasB method in detection water according to claim 1, it is characterised in that step Comprising the following steps that for sample DNA is extracted described in rapid A：50mL water samples 10000rpm is centrifuged 10 minutes, or takes 50mL water samples to be existed by membrane filtration with tweezers gripping filter membrane In 50mL centrifuge tube, with 10000rpm is centrifuged 10 minutes again after 2ml ultrapure waters, supernatant is removed；Add 5mL CTAB extractions Liquid, fully mix.65 DEG C of incubation 30min, vibrate frequently；Afterwards 8000rpm room temperatures centrifugation 10min, take 1ml supernatants enter 2ml from In heart pipe；Forced oscillation, 13000rpm centrifugations 10min, the transfer μ L of supernatant 600 to new 2ml centrifugations are used after adding 700 μ L chloroforms Guan Zhong；Adding 350 μ L NaCl solutions precipitation is suspended, add 350 μ L chloroforms, vortex oscillation is mixed, 13000rpm centrifuges 10min, and the isopropanol that 0.8 times of volume is added after transfer supernatant is used for precipitate nucleic acids, and room temperature places 20min, 13000rpm centrifuges 10min, supernatant discarding, adds the ethanol solutions of 500 μ L 70% washing precipitation, is dissolved in 50 μ L TE solution In.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913541A (en) * | 2021-11-29 | 2022-01-11 | 拱北海关技术中心 | Primer, probe and kit for rapidly detecting pseudomonas aeruginosa in food |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429539A (en) * | 2007-11-09 | 2009-05-13 | 中山大学达安基因股份有限公司 | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus |
| CN105950732A (en) * | 2016-05-25 | 2016-09-21 | 中国农业大学 | Animal-derived food pathogen identification and drug-resistant and toxic gene detection composite chip |
| CN106048052A (en) * | 2016-07-29 | 2016-10-26 | 中山出入境检验检疫局检验检疫技术中心 | Kit and method for detecting staphylococcus aureus and integron in food |
| CN106520984A (en) * | 2016-12-06 | 2017-03-22 | 湖南圣湘生物科技有限公司 | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method |
-
2017
- 2017-07-17 CN CN201710579934.9A patent/CN107419012A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101429539A (en) * | 2007-11-09 | 2009-05-13 | 中山大学达安基因股份有限公司 | Reagent kit for real time fluorescence quantitative PCR detection of bacillus pyocyaneus |
| CN105950732A (en) * | 2016-05-25 | 2016-09-21 | 中国农业大学 | Animal-derived food pathogen identification and drug-resistant and toxic gene detection composite chip |
| CN106048052A (en) * | 2016-07-29 | 2016-10-26 | 中山出入境检验检疫局检验检疫技术中心 | Kit and method for detecting staphylococcus aureus and integron in food |
| CN106520984A (en) * | 2016-12-06 | 2017-03-22 | 湖南圣湘生物科技有限公司 | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| FATEMEH FARAJI等: "Molecular detection of virulence genes in Pseudomonas aeruginosa isolated from children with Cystic Fibrosis and burn wounds in Iran", 《MICROBIAL PATHOGENESIS》 * |
| 宋月等: "绿脓杆菌SYBR Green I 实时定量PCR检测方法的建立及初步应用", 《华北农学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913541A (en) * | 2021-11-29 | 2022-01-11 | 拱北海关技术中心 | Primer, probe and kit for rapidly detecting pseudomonas aeruginosa in food |
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