CN106148534A - For detecting shigella and the test kit of integron and method in food - Google Patents
For detecting shigella and the test kit of integron and method in food Download PDFInfo
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- CN106148534A CN106148534A CN201610614027.9A CN201610614027A CN106148534A CN 106148534 A CN106148534 A CN 106148534A CN 201610614027 A CN201610614027 A CN 201610614027A CN 106148534 A CN106148534 A CN 106148534A
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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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q2600/16—Primer sets for multiplex assays
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Abstract
The invention discloses and a kind of detect shigella and the primer of integron, probe, test kit and method in food, the sequence of primer is: described in forward primer SHGF:AAGACCAAAGAGGGGGACCT downstream primer SHGR:TGTCTCAGTTCCAGTGTGGC, the sequence of integron primer is: forward primer int1F:CATCGTCGTAGAGACGTCGG downstream primer Int1R:AGAACAAGCAGGCATCACGA.Primer, probe and other component, reasonable mixture ratio in the test kit of the present invention, easy to use, detection is quick, accurately, it is adaptable to shigella and integron int1 in dual ddPCR detection food；Detection method is easy and simple to handle, quick, low cost, and testing result is accurate.
Description
Technical field
The present invention relates to a kind of detect in food and the primer of integron int1, and with this primer with the use of spy
Pin；The invention still further relates to a kind of dual droplet type digital pcr detection kit comprising above-mentioned primer and probe, the present invention also relates to
And a kind of use mentioned reagent box detection food in shigella and the method for integron int1.
Background technology
Shigella Shigella, for gram negative bacilli, in arcuation, the various shape such as shaft-like, thread, anodontia spore.
The feed food containing this bacterium can cause alimentary toxicosis, also referred to as halophilic bacteria alimentary toxicosis.Shigella is widely present in all kinds of life
Or vegetable for processing, milk and milk product, fowl, meat products, fruit, bread product, hamburger etc..Shigella Fast Detection Technique is built
Stand and will improve food-borne pathogens detection efficiency and detection flux, for testing agency's offer technical support of different condition, meet
In food, the quick testing requirement of shigella, preferably ensures food safety, and the most domestic and international Trade Development, at food hygiene
Significant with food safety detection aspect.It is contemplated that set up in a kind of detection animal derived food quick, accurate
Shigella method, improves the detection efficiency of food-borne shigella, provides skill for the monitoring of animal derived food pathogenic microorganism
Art means.
1989, Strokes proposed the concept of integron first, and within 1991, Hall formally proposes box gene-integron
The concept of system.Integron is the DNA fragmentation of antibacterial, closely related with bacterial drug resistance, be one be present in bacterial plasmid or
Genetic elements system on chromosome, is obtained exogenous gene by the intergrase of self coding and is allowed to express.Integron is caught
Obtain, integrate and express drug resistance gene box, cause drug resistant gene to propagate between Pseudomonas of the same race or the most of the same race, so that antibacterial is resistance to
The property of medicine is able to extensive diffusive.Box gene is by being sheared down during on integron, intergrase is integrated into integron or from integron
Come, make drug resistant gene be propagated and diffusion.
At present, outside existing 130 multidrug resistant gene boxes are identified that integron passes through locus specificity restructuring capture and expresses
Source box gene, thus give Host Strains and various common drug kinds are produced drug resistance.Integron self can be located at joint simultaneously
On the mobility elements such as character grain, transposon, whole integron can be made to be moved.Thus integron is in new Resistant strain
Appearance and drug resistant gene level send out in play very important role.Integron is according to the primary structure of integrase protein
It is broadly divided into 4 classes, but class 1 integron is most common.The present invention by design for shigella and the special primer of integron,
Probe combinations, it may be appreciated that the distribution of shigella and the popularity of integron in bacterial strain in different food products.
At present, varieties of food items safety criterion is desirable that and shigella carries out detection by quantitative, but for shigella
Detection by quantitative is counted after mainly or being cultivated by traditional method.And conventional PCR method carries out electricity after needing PCR amplification
Swimming, not only complex operation, and detection by quantitative can not be realized.At present, Southern blot and real-time fluorescence quantitative PCR are
Two kinds of conventional copy number of foreign gene analytical technologies, are widely used in copy number of foreign gene analysis.But both approaches is also
There is certain defect.Such as, during Southern blot methods analyst, workload is big, cycle length, operation require that high, accuracy is relatively
Difference, especially for the analysis of multi-copy gene, result is less than normal.QRT-PCR is necessary when analyzing copy number of foreign gene
Depending on standard curve and the gene of known copy number, simply one relative quantitation method, and the quality of standard curve is vulnerable to
The factors impacts such as the concentration of DNA purity, primer and probe, the response inhabitation factor；It addition, standard curve must be based on standard
Material is set up, and the limitednumber of standard substance and expensive price are not applied for all of research.
Microdroplet digital pcr (droplet digital PCR, ddPCR) is a kind of new absolute quantitation of rising in recent years
Technology, it carries out the nucleic acid quantification counted, is a kind of method of absolute quantitation based on single-molecule PCR method.Main employing is worked as
Micro-fluidic or the microdroplet method of front analytical chemistry hot topic research field, is dispersed to chip by the nucleic acid solution after Macrodilution
In microreactor or microdroplet, the nucleic acid-templated number of each reactor is less than or equal to 1.So after PCR cycle, have
The reactor of one nucleic acid templates will provide fluorescence signal, does not has the reactor of template just not have fluorescence signal.According to
Relative scale and the volume of reactor, it is possible to extrapolate the gene copy number of original solution.
But the analysis work of copy number based on microdroplet digital pcr platform report is less, for shigella and integron
Detection the most more have no and report.The present invention, based on droplet type ddPCR platform, establishes shigella and integron genes is copied
Shellfish number analyze method, result of study be analyze food safety biogenic risks and assumptions detection by quantitative provide new method and
Use for reference, also provide technical support means for food safety quality control.
Summary of the invention
The invention aims to overcome weak point of the prior art, it is provided that one is used for detecting will in food and congratulates
Salmonella and the primer of integron int1, and with this primer with the use of probe；
It is a further object to provide a kind of test kit comprising above-mentioned primer and probe, drawing in this test kit
Thing, probe and other component, reasonable mixture ratio, easy to use, detection is quick, accurately, it is adaptable in dual ddPCR detection food
Shigella and integron int1；
Another object of the present invention is to provide shigella and integron in a kind of employing mentioned reagent box detection food
The method of int1, the method is easy and simple to handle, quick, low cost, and testing result is accurate.
In order to achieve the above object, the present invention uses below scheme:
A kind of detect shigella and the primer of integron in food, it is characterised in that include shigella primer and whole
Zygote primer；
The sequence of described shigella primer is:
Forward primer SHGF:AAGACCAAAGAGGGGGACCT
Downstream primer SHGR:TGTCTCAGTTCCAGTGTGGC
The sequence of described integron primer is:
Forward primer int1F:CATCGTCGTAGAGACGTCGG
Downstream primer Int1R:AGAACAAGCAGGCATCACGA.
The present invention and primer described above with the use of probe, it is characterised in that include shigella probe and integration
Sub-probe:
The sequence of described shigella probe is:
Probe SHGP:TAGCTGGTCTGAGAGGATGA
The sequence of described integron probe is:
Probe I nt1P:AGGGTGTGCGGTGTGGCGGGC.
The present invention is for detecting shigella and the test kit of integron in food, it is characterised in that comprise claim 1
In primer and probe in claim 2.
It is previously discussed for detecting shigella and the test kit of integron in food, it is characterised in that in this test kit
20.0 μ L reaction systems include following components: 2 × ddPCR Super Mix10.0 μ L, shigella and integron int1 are just
The each 1.0 μ L of reverse primer are or/and each 1.0 μ L of the probe of shigella and integron int1, DNA profiling 4.0 μ L.
For detecting shigella and the method for integron in food, it is characterised in that comprise the following steps:
A, extraction sample DNA；
B, by each reactive component add 20.0 μ l reaction systems as claimed in claim 4, be subsequently adding 70.0 μ l mineral
Oil, transfers to automatically generate on drop generator microdroplet after mixing；Take the positive quality control in test kit and negative matter the most respectively
Control, prepares corresponding DNA profiling；
C, digital pcr mixed liquor ddPCR Super Mix is fabricated to the micro-reaction system of Water-In-Oil PCR, and all shifts
In 96 hole Sptting plate PCR reaction tubes；Again by 96 hole Sptting plate PCR reaction tubes at sealer instrument upper sealing film, it is placed in regular-PCR instrument
Carry out PCR reaction；
D, opening microdroplet fluorescence detector application software, 96 hole Sptting plate PCR reaction tubes after PCR reaction being terminated are direct
Insertion equipment, detects the PCR response situation of microdroplet in each 96 hole Sptting plate PCR reaction tubes, finally according to amber pine distribution law
Calculate the copy number of testing gene.
It is previously discussed for detecting shigella and the method for integron in food, it is characterised in that PCR amplification in step C
Response procedures sequentially include the following steps:
(1) 94 DEG C of denaturation 3min；
(2) 94 DEG C of degeneration 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 of stopped reaction.
It is previously discussed for detecting shigella and the method for integron in food, it is characterised in that carry described in step A
Take specifically comprising the following steps that of sample DNA
Take 1.5mL culture 10000rpm and be centrifuged 2min；Precipitate adds the TE buffer of 500 μ L, and piping and druming is allowed to repeatedly
Eddy diffusion, adds 30 μ L 10%SDS, and mixing, in 37 DEG C of incubation 1h；Add 100 μ L 5mol/L NaCl, fully mix, then
Add 80ul CTAB/NaCl solution, 65 DEG C of incubation 10min again after mixing；Add isopyknic phenol/chloroform/isoamyl alcohol mixing,
Centrifugal 4-5min, proceeds in a new pipe by supernatant, adds the isopropanol of 0.8 times of volume, is gently mixed under precipitating until DNA
Come；After 70% washing with alcohol of precipitation 1mL, add TE solution dissolution precipitation.
It is previously discussed for detecting shigella and the method for integron in food, it is characterised in that step B takes the positive
The method that Quality Control prepares corresponding DNA profiling with negative Quality Control is identical with the method in step A.
Sensitivity and Specificity test in the present invention:
Using the methods such as order-checking that positive amplification product carries out sequence verification, result positive amplification Product Sequence is carried out
When Blast compares, sequence all with Genbank aim sequence very high homology.10 times of reference strain genomic DNAs diluted are added
Previous reaction system, repeats have fine repeatability in test display detection sample.Dilute template concentrations logarithm value and copy
Also in good linear relationship R between shellfish numerical value2≥0.95.Illustrate that the method has preferable degree of accuracy and good stablizing
Property.
The present invention compared with prior art, has the advantage that
1) reagent constituents of the present invention and reasonable mixture ratio, easy to use, and detection is quick, accurately, it is adaptable to ddPCR quantitatively examines
Survey shigella and integron (int1)；
2) detection method simplifies testing process, and without making standard curve, substantially reduces detection week
Phase, the detection time cultivates method of counting than tradition and shortens about two days；
3) the whole process of detection method is without using standard curve, and direct with new-generation sequencing slitless connection,
Gene copy number can be performed absolute quantification analysis.
4) digital pcr detecting system is processed by microdroplet, can greatly reduce the interference of background and substrate, and sensitivity can
With as little as 1 copy, therefore, the slight change to low concentration mrna concentration carries out accurate and repeated good detection.
5) use detection method can realize shigella and integron simultaneously in same reaction system and carry out essence
Quasi-detection, easy and simple to handle, quick.There is preferable industrialization prospect.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described further:
Embodiment 1
The present invention detects shigella and the primer of integron int1 and probe in food, and the sequence of this primer and probe is divided
It is not:
Forward primer SHGF:AAGACCAAAGAGGGGGACCT
Downstream primer SHGR:TGTCTCAGTTCCAGTGTGGC
Probe SHGP:TAGCTGGTCTGAGAGGATGA
Forward primer int1F:ATGATCGTGCCGTGATCGAA
Downstream primer Int1R:AGCTTCTGTATGGAACGGGC
Probe I nt1P:GCAAACCCTCACTGATCCGC
Embodiment 2
The present invention is a kind of detects shigella and the test kit of integron int1 in food, and wherein 20 μ L reaction systems include
Following components:
The each 1.0 μ L of 2 × ddPCR Super Mix 10.0 μ L, shigella and (int1) forward and reverse primer, probe each 1.0
μ L, DNA profiling 4.0 μ L.
Wherein primer sequence is as follows:
Forward primer 1, SHGF:AAGACCAAAGAGGGGGACCT
Downstream primer 1, SHGR:TGTCTCAGTTCCAGTGTGGC
Probe 1, SHGP:TAGCTGGTCTGAGAGGATGA
Forward primer 2, int1F:ATGATCGTGCCGTGATCGAA
Downstream primer 2, Int1R:AGCTTCTGTATGGAACGGGC
Probe 2, Int1P:GCAAACCCTCACTGATCCGC.
Embodiment 3
The present invention detects shigella and the method for integron (int1) in food, including following
Step:
A, extraction sample DNA；
B, by each reactive component add above-mentioned 20.0 μ l reaction systems, be subsequently adding 70.0 μ l mineral oil, after mixing shift
Microdroplet is automatically generated on drop generator；
C, the microdroplet of generation is carefully transferred completely in 96 hole Sptting plate PCR reaction tubes；Again by 96 hole Sptting plates in envelope
Film instrument upper sealing film, is placed in regular-PCR instrument and carries out PCR reaction.
D, opening microdroplet fluorescence detector application software, 96 hole Sptting plates after PCR reaction being terminated are inserted directly into equipment,
Detect the PCR response situation of microdroplet in every PCR reaction tube, calculate the copy number of testing gene finally according to amber pine distribution law.
Wherein the sequence of primer and probe is as follows:
Forward primer SHGF:AAGACCAAAGAGGGGGACCT
Downstream primer SHGR:TGTCTCAGTTCCAGTGTGGC
Probe SHGP:TAGCTGGTCTGAGAGGATGA
Forward primer int1F:ATGATCGTGCCGTGATCGAA
Downstream primer Int1R:AGCTTCTGTATGGAACGGGC
Probe I nt1P:GCAAACCCTCACTGATCCGC.
Described fluorescent PCR amplification sequentially includes the following steps:
(1) 94 DEG C of denaturation 3min；
(2) 94 DEG C of degeneration 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 of stopped reaction.
Embodiment 4
Increase bacterium sample before weighing 1g, wherein can adjust sample size according to the DNA content of sample, join the centrifugal of 50mL
Guan Zhong；Add 5mL extract with CTAB liquid, fully mix.Hatch 30min, frequently vibrate for 65 DEG C；The food samples that water absorption is big, adds
Enter extract with CTAB liquid amount should according to liquid level whether can cover sample and have more a little depending on；65 DEG C overnight after, 8000r/min room
The centrifugal 10min of temperature, takes 1ml supernatant and enters in 2ml centrifuge tube；Using forced oscillation after adding 700 μ L chloroforms, 13000g is centrifuged 10min,
Transfer supernatant 600 μ L is in new 2ml centrifuge tube；Adding the CTAB precipitation solution of 2 times of volumes, reverse rear chamber is gentle and quiet for several times puts
60min, 13000 × g are centrifuged 10min, supernatant discarded, add 350 μ L NaCl solution and precipitation are suspended, add 350 μ L
Chloroform, vortex oscillation mixes, and 13000g is centrifuged 10min, and the isopropanol adding 0.8 times of volume after transfer supernatant is used for precipitating
Nucleic acid, room temperature is placed 20min, 13000g and is centrifuged 10min, supernatant discarded, adds 500 μ L 70% ethanol solution washing precipitations, molten
Solution is in 50 μ L TE solution.Then take 4.0 μ L extracts and join following reaction system,
The each 1.0 μ L of 2 × ddPCR Super Mix 10.0 μ L, shigella and (int1) forward and reverse primer, probe each 1.0
μ L, DNA profiling 4.0 μ L.
Wherein the sequence of primer and probe is as follows:
Forward primer SHGF:AAGACCAAAGAGGGGGACCT
Downstream primer SHGR:TGTCTCAGTTCCAGTGTGGC
Probe SHGP:TAGCTGGTCTGAGAGGATGA
Forward primer int1F:ATGATCGTGCCGTGATCGAA
Downstream primer Int1R:AGCTTCTGTATGGAACGGGC
Probe I nt1P:GCAAACCCTCACTGATCCGC.
Carry out PCR amplification, sequentially include the following steps:
(1) 94 DEG C of denaturation 3min；
(2) 94 DEG C of degeneration 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 of stopped reaction.
In order to prove the accuracy of detection method, the present invention has made comparative testing below: be respectively adopted the present invention
" national food safety standard food microbiological examination shigella is examined for detection method and colony counting method GB 4789.5-2012
Test " carry out the comparison of shigella quantitative measurement.Concrete experimental implementation is all carried out according to standard test numerical value, tests repetition three
Secondary, results averaged.Result is as shown in table 1, from the results, it was seen that the present invention uses digital pcr method to big in food
The measurement result of intestinal flora quantity, compared with existing GB 4789.5-2012,10 parts of samples, mean error is 1.9%, this explanation
The inventive method has the highest accuracy.It addition, digital pcr of the present invention is enjoyed oneself to the full, single sample whole process detection time of method is 4
Hour, method of counting cultivated by the most existing traditional flat board, and while detecting shigella, moreover it is possible to its integron is entered
Row well detection, it is possible to accurately Fast Evaluation food-borne pathogens and pathogenic bacterium carry the propagation risk of drug resistant gene.Therefore
There is good technical advantage and Developmental Prospect of Industrialization.
The ultimate principle of the present invention and principal character and advantages of the present invention have more than been shown and described.The skill of the industry
The art personnel simply explanation it should be appreciated that the present invention is not restricted to the described embodiments, described in above-described embodiment and description
The principle of the present invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, these
Changes and improvements both fall within scope of the claimed invention.Claimed scope by appending claims and
Its equivalent defines.
Claims (8)
1. one kind is detected shigella and the primer of integron in food, it is characterised in that include shigella primer and integration
Sub-primer；
The sequence of described shigella primer is:
Forward primer SHGF:AAGACCAAAGAGGGGGACCT
Downstream primer SHGR:TGTCTCAGTTCCAGTGTGGC
The sequence of described integron primer is:
Forward primer int1F:CATCGTCGTAGAGACGTCGG
Downstream primer Int1R:AGAACAAGCAGGCATCACGA.
2. with claim 1 described in primer with the use of probe, it is characterised in that include shigella probe and integron
Probe:
The sequence of described shigella probe is:
Probe SHGP:TAGCTGGTCTGAGAGGATGA
The sequence of described integron probe is:
Probe I nt1P:AGGGTGTGCGGTGTGGCGGGC.
3. for detecting the test kit of shigella and integron, it is characterised in that comprise the primer in claim 1 and right
Require the probe in 2.
The most according to claim 3 for detecting the test kit of shigella and integron, it is characterised in that in this test kit
20.0 μ L reaction systems include following components: 2 × ddPCR Super Mix10.0 μ L, shigella and integron int1 are just
The each 1.0 μ L of reverse primer are or/and each 1.0 μ L of the probe of shigella and integron int1, DNA profiling 4.0 μ L.
5. for detecting shigella and the method for integron in food, it is characterised in that comprise the following steps:
A, extraction sample DNA；
B, each reactive component is added 20.0 μ l reaction systems as claimed in claim 4, be subsequently adding 70.0 μ l mineral oil, mixed
Transfer to automatically generate on drop generator microdroplet after even；Take the positive quality control in test kit and negative Quality Control, system the most respectively
Standby corresponding DNA profiling；
C, digital pcr mixed liquor ddPCR Super Mix is fabricated to the micro-reaction system of Water-In-Oil PCR, and is transferred completely into 96
In the Sptting plate PCR reaction tube of hole；Again by 96 hole Sptting plate PCR reaction tubes at sealer instrument upper sealing film, it is placed in regular-PCR instrument and carries out
PCR reacts；
D, opening microdroplet fluorescence detector application software, 96 hole Sptting plate PCR reaction tubes after PCR reaction being terminated are inserted directly into
Equipment, detects the PCR response situation of microdroplet in each 96 hole Sptting plate PCR reaction tubes, calculates finally according to amber pine distribution law
The copy number of testing gene.
The most according to claim 5 for detecting shigella and the method for integron in food, it is characterised in that in step C
The response procedures of PCR amplification sequentially includes the following steps:
(1) 94 DEG C of denaturation 3min；
(2) 94 DEG C of degeneration 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 of stopped reaction.
The most according to claim 5 for detecting shigella and the method for integron in food, it is characterised in that in step A
Specifically comprising the following steps that of described extraction sample DNA
Take 1.5mL culture 10000rpm and be centrifuged 2min；Precipitate adds the TE buffer of 500 μ L, and piping and druming is allowed to again repeatedly
Suspending, add 30 μ L 10%SDS, mixing, in 37 DEG C of incubation 1h；Add 100 μ L 5mol/L NaCl, fully mix, add
80ul CTAB/NaCl solution, 65 DEG C of incubation 10min again after mixing；Add isopyknic phenol/chloroform/isoamyl alcohol mixing, centrifugal
4-5min, proceeds in a new pipe by supernatant, adds the isopropanol of 0.8 times of volume, is gently mixed until DNA precipitates；Heavy
After 70% washing with alcohol of shallow lake 1mL, add TE solution dissolution precipitation.
The most according to claim 7 for detecting shigella and the method for integron in food, it is characterised in that in step B
Take positive quality control identical with the method in step A with the method that corresponding DNA profiling is prepared in negative Quality Control.
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| CN107419016A (en) * | 2017-07-17 | 2017-12-01 | 蔡先全 | Detect the primer of pseudomonas aeruginosa and integron, kit and method in water |
| CN108220401A (en) * | 2018-03-16 | 2018-06-29 | 张家港出入境检验检疫局检验检疫综合技术中心 | A kind of campylobacter jejuni recombinant plasmid standard items quantitative detecting method based on droplet type digital pcr |
| CN109762917A (en) * | 2019-03-28 | 2019-05-17 | 广东省实验动物监测所 | Multiple liquid phase genetic chip primer, kit and its analysis method a kind of while that detect 5 Sulfonamides-resistant genes |
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