CN106244691A - A kind of based on the method for Salmonella in microdroplet digital pcr method fast quantification examination Fructus Fragariae Ananssae - Google Patents
A kind of based on the method for Salmonella in microdroplet digital pcr method fast quantification examination Fructus Fragariae Ananssae Download PDFInfo
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- CN106244691A CN106244691A CN201610632540.0A CN201610632540A CN106244691A CN 106244691 A CN106244691 A CN 106244691A CN 201610632540 A CN201610632540 A CN 201610632540A CN 106244691 A CN106244691 A CN 106244691A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
The invention discloses a kind of based on the method for Salmonella in microdroplet digital pcr method fast quantification examination Fructus Fragariae Ananssae, the method is with Salmonella invA virulence gene synthesis specific primer and probe, extracting DNA of bacteria in Fructus Fragariae Ananssae by Chelex 100 method, application microdroplet digital pcr technology carries out fast quantification examination to Salmonella.The method that the present invention is set up is without building plasmid and standard curve, avoid and affect the accuracy of sample definite value because of standard curve value deviation, whole testing process can complete the overall process of sample in 4 hours and identify, having the advantages such as efficient, quick, real-time, easy and simple to handle, in fresh fruit vegetables Fructus Fragariae Ananssae of making a living, the fast quantification examination of Salmonella provides technical support.
Description
Technical field
The invention belongs to food microorganisms identify and applied technical field, be specifically related to Salmonella in fresh fruit and vegerable Fructus Fragariae Ananssae
Bacterium, DNA extraction and microdroplet digital pcr fast quantification screening method.
Background technology
Instant raw vegetable and fruit are one of main carriers of food-borne pathogens propagation.At present, State Administration of Quality Supervision, Inspection and Quarantine will eat
The Risk Monitoring of product safety has listed the great solution problem of people's livelihood people meter in, affect food safety because have a lot, and wherein
The food-safety problem caused by harms of microbe has reached the 40% of total amount[1-2].China is in the micro-life of foodborne pathogens
Thing aspect is faced with stern challenge, is badly in need of potential hazard is made rational risk assessment, regains the initiative and harm is controlled
System is at acceptable level[3].And the technological means of microorganism Quantitative Monitoring is mainly culture-based method at present, waste time and energy, auxiliary
Real time fluorescence quantifying PCR method, though there being very big breakthrough in time, but the accuracy of its quantitative result standard to be depended on
The accuracy of curve structure and the amplification efficiency of primer, and the application that microdroplet digital pcr technology is on foodborne pathogens, make up
This defect.
Microdroplet digital pcr technology, as third generation round pcr, uses a kind of brand-new on the basis of traditional PCR method
Mode carries out the quantitative of nucleic acid molecules, compared with real-time fluorescence quantitative PCR, it is not necessary to build standard curve, not by amplification efficiency
Impact, its result has higher degree of accuracy, accuracy and sensitivity.The core of microdroplet digital pcr is can be by a sample
It is divided into 20, the microdroplet of 000 nanoliter of level, the most each microdroplet or without nucleic acid target molecule to be checked or the most several containing
Nucleic acid target molecule to be checked, and each microdroplet is as an independent PCR reactor.After PCR expands, use microdroplet analyser
Detecting each microdroplet one by one, the microdroplet interpretation having fluorescence signal is 1, and the microdroplet interpretation not having fluorescence signal is 0, finally
According to Poisson distribution principle and the ratio of positive microdroplet, analyze software and can calculate concentration or the copy providing target molecule to be checked
Number, in the way of absolute quantitation, direct " number " goes out the number of target molecule[4]。
Salmonella is most important pathogen in Bacterium entericum, the Salmonella infection of people and carry disease germs the most universal.Food
Animal or food with infecting before death are contaminated, and Crinis Carbonisatus uncooked food all can be made to be poisoned.In alimentary toxicosis all over the world, husky
Door Salmonella alimentary toxicosis often accounts for first place or second.Culture-based method is to commonly use detection method at present, but its detection Salmonella
Testing procedure complexity is loaded down with trivial details, time and effort consuming, overall process needs at least 4～7d, just can draw clear and definite diagnostic result.Thus, build
The focus of vertical a kind of method always research detecting Salmonella quickly and accurately[5], and the application of microdroplet digital pcr solves
This difficult problem, and filled up the microdroplet digital pcr technology blank in foodborne pathogens detection.
Research shows, the invasin protein (invasion protein, inv) of Salmonella determines that antibacterial enters host epithelial
The ability of cell, pathogenic closely related with Salmonella, it is by one group of base such as invA, invB, invC, invD and invE
Because of coding, wherein invA is the Major Virulence Factors of Salmonella, therefore according to invA virulence base on the basis of forefathers study
Because of sequent synthesis specific primer and TaqMan probe, groped by the optimization of the test such as specificity, sensitivity, set up microdroplet number
Word PCR Salmonella fast quantitative measurement method for detecting, with its overcome tradition pathogenic microorganism detection method inconvenience, and with biography
System national standard method compares, and opens new technical field for foodborne pathogens detection.
Summary of the invention
Can be quick, easy when it is an object of the invention to the Salmonella in the large sample fresh Fructus Fragariae Ananssae of quantitative examination
Obtain screening results, spreading, when striving for effective for food safety accident of very first time control food-borne pathogens
Between.The quick screening method of the present invention has the advantages such as efficient, quick, accurate, easy and simple to handle.
For achieving the above object, the present invention provides following technical scheme:
A kind of based on the method for Salmonella in microdroplet digital pcr method fast quantification examination Fructus Fragariae Ananssae, it is characterised in that it
Carry out as follows:
(1) weigh Fructus Fragariae Ananssae sample 25g with reference to GB GB4789 method, add 225mL0.85% sterile saline, make
The diluent of 1:10, with the abundant homogenizing of homogenizer；
(2) take the diluent 100mL of 1:10, utilize the bacteria gathering device of sterilizing, the diluent of 100mL 1:10 is enriched in hole
Footpath is on the collection Mycoderma of 0.45 μm, extracts Fructus Fragariae Ananssae DNA of bacteria in collection Mycoderma by Chelex-100 method；
(3) use microdroplet digital pcr amplimer and probe primer, the DNA of Salmonella is expanded；
Microdroplet digital pcr reaction system therein is 20 μ L, and each component content is: 2 × ddPCR Supermix for
Probes premixed liquid 10 μ L, each 1.6 μ L of upstream and downstream primer, probe primer 0.4 μ L, template 2 μ L, aseptic ultra-pure water supplies 20 μ L,
It is centrifuged after mixing.It is carefully transferred to microcaloire generator, never produces bubble, add with microcaloire generator relevant position simultaneously
Heavy oil 70 μ L, covers pad, puts into generation microdroplet in drop generators.Draw the microdroplet that 40 μ L generate, be carefully transferred to numeral
In the Sptting plate of PCR96 hole, starting the cycle over after 170 DEG C of heat-sealings in PCR instrument, PCR instrument Ramp Rate is set to 2 DEG C/s, circulation ginseng
Number is: 95 DEG C, 10min；40cycles (94 DEG C, 30s, 60 DEG C, 1min)；98 DEG C, 10min.
Fast quantification screening method of the present invention, Chelex-100 method extracts Fructus Fragariae Ananssae DNA of bacteria in collection Mycoderma, wherein
Chelex-100 solution concentration is 13%.
In order to enable more clearly to illustrate the quick screening method of the present invention, real with fresh fruit and vegerable Fructus Fragariae Ananssae for sample below
Example, is illustrated the quick screening method of the present invention.
One, material and primer
(1) sample: Fructus Fragariae Ananssae sample, is purchased by the market of farm produce.
(2) key instrument: High speed refrigerated centrifuge, microdroplet digital pcr instrument, thermostat water bath.
(3) main agents:
PCR primer, probe are synthesized by Shanghai Sheng Gong company, and primer sequence is shown in Table 1.
2 × ddPCR Supermix for probes premixed liquid, reagent is purchased from Bole company.
Table 1 Salmonella identifies quantification PCR primer sequence
Two, in fresh fruit and vegerable Fructus Fragariae Ananssae, DNA of bacteria is extracted
(1) weigh Fructus Fragariae Ananssae sample 25g with reference to GB GB4789 method, add 225mL0.85% sterile saline, make
The diluent of 1:10, with the abundant homogenizing of homogenizer；
(2) take the diluent 100mL of 1:10, utilize the bacteria gathering device of sterilizing, the diluent of 100mL 1:10 is enriched in hole
Footpath is on the collection Mycoderma of 0.45 μm；
(3) Fructus Fragariae Ananssae DNA of bacteria in collection Mycoderma is extracted by Chelex-100 method.
Three, the quantitative Screening tests of microdroplet digital pcr of Salmonella DNA in Fructus Fragariae Ananssae:
1, PCR reaction system composition:
2 × ddPCR Supermix for probes premixed liquid 10 μ L, each 1.6 μ L of upstream and downstream primer, probe primer 0.4 μ
L, template 2 μ L, aseptic ultra-pure water supplies 20 μ L, centrifugal after mixing.
2, microdroplet generates:
It is carefully transferred to microcaloire generator, never produces bubble, add weight with microcaloire generator relevant position simultaneously
Oil 70 μ L, cover pad, put into generation microdroplet in drop generators.Draw the microdroplet that 40 μ L generate, be carefully transferred to numeral
In the Sptting plate of PCR96 hole, expanding in PCR instrument after 170 DEG C of heat-sealings, PCR instrument Ramp Rate is set to 2 DEG C/s.
3, PCR response procedures:
95 DEG C of denaturations 10min；40 amplification cycles (94 DEG C, 30sec；60 DEG C, 1min)；98 DEG C, 10min.
4, microdroplet digital pcr amplification:
By copy number, calculating Fructus Fragariae Ananssae sample Salmonella concentration, result is shown in accompanying drawing 1.Amplification and culture-based method
Qualification result is basically identical.
What the present invention had has the active effect that
(1) microdroplet digital pcr endpoint quantification method is used, effectively overcome because traditional method is time-consuming, laborious, loaded down with trivial details,
And detection excessive cycle, it is difficult to meet the needs that emergency preplan processes.
(2) advantage that this method is maximum is that its process is quick, easy, precisely, substantially increases in fresh fruit and vegerable Fructus Fragariae Ananssae
The detection efficiency that Salmonella is identified.Compared with the time that traditional culture identification process needs nearly 1 week, employing this method, 4
Hour can complete the overall process of sample examination.
(3) use the inventive method that Fructus Fragariae Ananssae sample carries out quantitative examination, disposably the sample size of examination can compare tradition
Culture method is greatly improved.
Accompanying drawing illustrates:
Fig. 1 is Salmonella microdroplet digital pcr AFLP system in Fructus Fragariae Ananssae sample；It is respectively positive control as schemed mark；Empty
White comparison；Fructus Fragariae Ananssae sample；
Fig. 2 is Salmonella microdroplet digital pcr AFLP system in cream strawberry sample；As the most positive right in schemed mark
According to；Blank；Cream strawberry sample；
Fig. 3 is Salmonella microdroplet digital pcr AFLP system in Charlie's Fructus Fragariae Ananssae sample；As the most positive right in schemed mark
According to；Blank；Charlie's Fructus Fragariae Ananssae sample；
Detailed description of the invention
In order to enable the method that the present invention is more clearly described, below in conjunction with embodiment, the present invention is further retouched
State.
Embodiment 1
With cream strawberry as sample, use the inventive method fast quantification examination Salmonella.Preparation method is as follows:
(1) weigh Fructus Cucumidis sativi sample 25g, carry out selective enrichment according to GB GB 4789 each pathogenic bacterium detection method；
(2) Salmonella after selective enrichment, staphylococcus aureus, Escherichia coli O 157: H7 and single increasing Lee are drawn
The each 1mL of enrichment liquid of this special Salmonella is in 2mL centrifuge tube, and 13200rpm is centrifuged 10min, abandons supernatant；Precipitation be dissolved in 100 μ L go from
In sub-water, sealed membrane is sealed, boiling water bath 10min；Quick freeze 10min in-20 DEG C of refrigerators after taking-up；Remove sealed membrane,
13200rpm is centrifuged 3min, and supernatant is DNA.
Real-time PCR detection:
1, four kinds of food-borne pathogens being carried out real-time fluorescent PCR amplification, primer sequence is:
SM-F:5 '-CTCACCAGGAGATTACAACATGG-3 '
SM-R:5 '-AGCTCAGACCAAAAGTGACCATC-3 '
SM-P:5 '-FAM-CACCGACGGCGAGACCGACTTT-TAMRA-3 '
2, reaction system:
2 × ddPCR Supermix for probes premixed liquid 10 μ L, each 1.6 μ L of upstream and downstream primer, probe primer 0.4 μ
L, template 2 μ L, aseptic ultra-pure water supplies 20 μ L, centrifugal after mixing.
3, response procedures:
95 DEG C of denaturations 10min；40 amplification cycles (94 DEG C, 30sec；60 DEG C, 1min)；98 DEG C, 10min.
4, microdroplet digital pcr amplification:
By copy number, calculating Salmonella concentration in cream strawberry sample, result is shown in accompanying drawing 2.
Embodiment 2
With Charlie Fructus Fragariae Ananssae as sample, use the inventive method fast quantification examination Salmonella.Preparation method is as follows:
(1) weigh Fructus Cucumidis sativi sample 25g, carry out selective enrichment according to GB GB 4789 each pathogenic bacterium detection method；
(2) Salmonella after selective enrichment, staphylococcus aureus, Escherichia coli O 157: H7 and single increasing Lee are drawn
The each 1mL of enrichment liquid of this special Salmonella is in 2mL centrifuge tube, and 13200rpm is centrifuged 10min, abandons supernatant；Precipitation be dissolved in 100 μ L go from
In sub-water, sealed membrane is sealed, boiling water bath 10min；Quick freeze 10min in-20 DEG C of refrigerators after taking-up；Remove sealed membrane,
13200rpm is centrifuged 3min, and supernatant is DNA.
Real-time PCR detection:
1, four kinds of food-borne pathogens being carried out real-time fluorescent PCR amplification, primer sequence is:
SM-F:5 '-CTCACCAGGAGATTACAACATGG-3 '
SM-R:5 '-AGCTCAGACCAAAAGTGACCATC-3 '
SM-P:5 '-FAM-CACCGACGGCGAGACCGACTTT-TAMRA-3 '
2, reaction system:
2 × ddPCR Supermix for probes premixed liquid 10 μ L, each 1.6 μ L of upstream and downstream primer, probe primer 0.4 μ
L, template 2 μ L, aseptic ultra-pure water supplies 20 μ L, centrifugal after mixing.
3, response procedures:
95 DEG C of denaturations 10min；40 amplification cycles (94 DEG C, 30sec；60 DEG C, 1min)；98 DEG C, 10min.
4, microdroplet digital pcr amplification:
By copy number, calculating Salmonella concentration in Charlie's Fructus Fragariae Ananssae sample, result is shown in accompanying drawing 3.
Claims (3)
1. for identifying pcr amplification primer thing and the probe primer of Salmonella, it is characterised in that include Salmonella primer forward
Sequence: 5 '-CTCACCAGGAGATTACAACATGG-3 ', reverse sequence: 5 '-AGCTCAGACCAAAAGTGACCATC-3 ', visit
Pin sequence: 5 '-FAM-CACCGACGGCGAGACCGACTTT-TAMRA-3 '.
2. one kind based on the method for Salmonella in microdroplet digital pcr method fast quantification examination Fructus Fragariae Ananssae, it is characterised in that it is pressed
Following steps are carried out:
(1) weigh Fructus Fragariae Ananssae sample 25g with reference to GB GB4789 method, add 225mL0.85% sterile saline, make 1:10
Diluent, with the abundant homogenizing of homogenizer；
(2) taking the diluent 100mL of 1:10, utilize the bacteria gathering device of sterilizing, the diluent of 100mL 1:10 is enriched in aperture is
On the collection Mycoderma of 0.45 μm, extract Fructus Fragariae Ananssae DNA of bacteria in collection Mycoderma by Chelex-100 method；
(3) use microdroplet digital pcr amplimer and probe primer, the DNA of Salmonella is expanded；
Microdroplet digital pcr reaction system therein is 20 μ L, and each component content is: 2 × ddPCR Supermix for probes
Premixed liquid Mix premixed liquid 10 μ L, upstream and downstream primer each 1.6 μ L, probe primer 0.4 μ L, template 2 μ L, aseptic ultra-pure water supplies 20 μ
L, centrifugal after mixing.It is carefully transferred to microcaloire generator, never produces bubble, add with microcaloire generator relevant position simultaneously
Enter heavy oil 70 μ L, cover pad, put into generation microdroplet in drop generators.Draw the microdroplet that 40 μ L generate, be carefully transferred to number
In the Sptting plate of word PCR96 hole, starting the cycle over after 170 DEG C of heat-sealings in PCR instrument, PCR instrument Ramp Rate is set to 2 DEG C/s, circulation
Parameter is: 95 DEG C, 10min；40cycles (94 DEG C, 30s, 60 DEG C, 1min)；98 DEG C, 10min.
3. Fructus Fragariae Ananssae DNA of bacteria during the Chelex-100 method described in claim 2 extracts collection Mycoderma, wherein Chelex-100 solution is dense
Degree is 13%.
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Cited By (3)
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| CN108085377A (en) * | 2017-12-29 | 2018-05-29 | 北京和益源生物技术有限公司 | The detection method of salmonella under a kind of high background |
| CN112649556A (en) * | 2020-11-13 | 2021-04-13 | 新疆大学 | Method for rapidly measuring concentration of hydrogen peroxide |
| CN114107530A (en) * | 2021-12-15 | 2022-03-01 | 上海交通大学 | Salmonella detection method and kit based on droplet digital PCR technology in lettuce |
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| CN114107530A (en) * | 2021-12-15 | 2022-03-01 | 上海交通大学 | Salmonella detection method and kit based on droplet digital PCR technology in lettuce |
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