CN108441568A - Detect the primer of C.perfringens and cpa genes, kit and method in food - Google Patents
Detect the primer of C.perfringens and cpa genes, kit and method in food Download PDFInfo
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Abstract
The invention discloses the primer of C.perfringens and Cpa genes, kit and methods in a kind of detection food, and the sequence and probe sequence of the primer are respectively:Sense primer recAF；Downstream primer recAR；Probe recAP；Sense primer CpaF；Downstream primer CpaR；Probe CpaP；Place that purpose of the invention is to overcome the shortcomings in the prior art, provides a kind of primer and component and proportioning are reasonable, and easy to use, detection is quick, accurately, is suitable for the kit of C.perfringens and Cpa dual ddPCR detection food；Another object of the present invention be to provide it is a kind of using mentioned reagent box detect food in the method for C.perfringens and Cpa, this method is easy to operate, quick, and testing result is accurate.
Description
Technical field
The present invention relates to the primers of C.perfringens and cpa genes in a kind of detection food, and the invention further relates to one kind
Including above-mentioned primer is detected for detecting the dual droplet type digital pcr (ddPCR) of C.perfringens and cpa genes in food
Kit detecting the method for C.perfringens and cpa genes in food food the invention further relates to a kind of using mentioned reagent box.
Background technology
C.perfringens (Clostridium perfringens) is a kind of gram-positive bacteria belonging to fusobacterium,
With pod membrane, gemma can be generated, does not have motility.C.perfringens be cause zoonosis the main pathogenic fungi it
One, the virulence factor of the bacterium is its generated exotoxin.At present, it has been found that the exotoxin more 17 that C.perfringens is generated
Kind, and it is most commonly seen with α types, β types, ε types and 4 kinds of toxin of ι types；C.perfringens also will produce other a variety of toxin or
The potential intoxicating factor, such as enterotoxin (CPE).According to the difference of institute's toxin producing, C.perfringens can be divided into 5 types.
The U.S. and some developing countries are the food poisoning of most common report by the microbial food poisonings of A types C.perfringens
One of.It poisons by food caused by C.perfringens mostly related to water, meat and birds meat products.Investigate the U.S. 1998-2010
Year by C.perfringens cause food poisoning the results show that meat product accounts for the 92% of food total amount, wherein beef product
46% is accounted for, birds meat products account for 30%, and pork product accounts for 16%.It is reported that fresh beef appetizer, pork, turkey meat and chicken and various
C.perfringens recall rates are more than 10% in meat products, and recall rate is even as high as 84% in the Japanese chicken in part.It is most common
During C.perfringens causes the faulty operation of food poisoning to betide food process and prepare, all factors are accounted for
93%.For example, by inappropriate cooling technique (44%) after meat products cooking；Meat products is positioned under improper reserve temperature
(22%).
16s rna genes have the stability of highly conserved and existing generality and nucleic acid sequence itself, sequence point
The reproducibility of analysis is high, and evolutionary rate is very slow, is referred to as " living fossil " of bacterium, the previous more use of detection bacterium quantity
All it is 16s RNA.But go deep into however as research, 16SrRNA genes also show the shortcomings that can not be ignored：Gao Bao
The species that keeping property keeps it closer from distinguishing affiliation well；Multicopy in genome makes the standard for determining its sequence
True property reduces.Therefore carry out more accurately taxonomic identification and and calculate some virulence gene copy number of bacterium when be difficult to really
It is fixed.The shortcomings that make up 16SrRNA genes, people begin attempt to using other different house-keeping genes (such as recA genes) into
The molecular biological analysis of row actinomyces.RecA is the central element of prokaryotes homologous recombination, is single copy gene, in DNA
It is a crucial gene during recombination and DNA damage lash-up recovering (SOS), is usually used in doing typing of bacteria.The present invention by its
House-keeping gene as C.perfringens carries out dual digital pcr and detects other virulence as its Molecular Identification gene
Gene effectively determines that bacterium is swollen to not only, but also an assessment can be carried out to virulence gene content.
Toxin (Clostridium perfringens alpha toxin, cpa) is considered as A type perfringens always
The main lethal toxins of clostridium.Cpa toxin is a kind of multi-function metal enzyme contained, dual with phosphatidase and sphingomyelinase
Activity.It can destroy the cell membrane being extremely made of phosphoric acid lecithin vinegar and phosphoric acid lecithin, and to by phosphatide phthalein serine, phosphatidic acid second
The cell membrane that hydramine, phosphatide phthalein glycerine form does not act on.In zoopery, by feeding on C.perfringens culture
Clear liquid can cause chicken that enteron aisle necrotic injury occurs.And chicken cannot be caused to send out after neutralizing the toxin in supernatant with toxin antibody
Raw intestinal tract injury.In view of above 2 points, for many years it has been recognized that cpa toxin is the principal causative for causing necrotic enteritis
The factor.
Since C.perfringens belongs to anaerobic bacteria, fostering requirement is higher, needs special anaerobic culture conditions and sets
Standby, in addition also needing to take particular sample mode, using corresponding culture medium, indentifying substance etc., general laboratory is difficult to carry out
Research to the bacterium, therefore the domestic effect to anaerobic bacteria and its in food poisoning for a long time also lacks understanding.In government and
The common people pay attention to today of food security, should assess the danger of anaerobic bacteria in food.
Conventional PCR method carries out electrophoresis after needing PCR amplification, not only cumbersome, but also can not be achieved quantitative detection.
Currently, Southern blot and real-time fluorescence quantitative PCR are common two kinds of copy number of foreign gene analytical technologies, extensively
It is analyzed for copy number of foreign gene.But there is also certain defects for both methods.For example, Southern blot methods are analyzed
When heavy workload, period be long, operation requires that high, accuracy is poor, especially for the analysis of multi-copy gene, be as a result easy inclined
It is small.Quantitative fluorescent PCR (qRT-PCR) is necessarily dependent upon standard curve and known copy number when analyzing copy number of foreign gene
Gene, only a kind of relative quantitation method, and the quality of standard curve are vulnerable to DNA purity, the concentration of primer and probe, reaction
The factors such as inhibiting factor influence；In addition, standard curve must be based on standard substance foundation, and the type of standard substance is limited
It is not applied for all research with expensive price.
Droplet digital pcr (droplet digital PCR, ddPCR) is a kind of new absolute quantitation of rising in recent years
Technology, the nucleic acid quantification that it is counted based on single-molecule PCR method are a kind of methods of absolute quantitation.By will be a large amount of
Nucleic acid solution after dilution is dispersed in the microreactor or droplet of chip, and the nucleic acid-templated number of each reactor is less than or waits
In 1.Pass through after PCR cycle in this way, there are one the reactors of nucleic acid templates will provide fluorescence signal, without template
Reactor just without fluorescence signal.According to the volume of relative scale and reactor, so that it may to extrapolate the gene of original solution
Copy number.
But the detection for C.perfringens recA and cpa gene just more has not been reported.The present invention is based on droplet types
DdPCR platforms, establish C.perfringens house-keeping gene recA and cpa gene copy number analysis method, and result of study is food
The quantitative detection of product safe biologic source property risks and assumptions provides new method.
Invention content
Place that purpose of the invention is to overcome the shortcomings in the prior art, provides a kind of primer and component and proportioning are closed
Reason, easy to use, detection is quick, accurately, the examination of C.perfringens and cpa genes food is detected suitable for dual ddPCR
Agent box；
Another object of the present invention is to provide a kind of detecting C.perfringens and cpa in food using mentioned reagent box
Method, 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：
The primer of C.perfringens and cpa genes in a kind of food, it is characterised in that the sequence and probe sequence of the primer
Row are respectively：
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The kit of C.perfringens and cpa genes in a kind of ddPCR detection food, it is characterised in that in the kit
20.0 μ L reaction systems include following components：2 × ddPCR Super Mix, 10.0 μ L, each 1.0 μ L of upstream and downstream primer, probe
Each 1.0 μ L, 4.0 μ L of DNA profiling.
Kit as described above, it is characterised in that the content ratio of the sense primer recAF and sense primer NetBF
Example is 0.1-1:1.
Kit as described above, it is characterised in that the content ratio of the downstream primer recAR and downstream primer NetBR
Example is 0.1-1:1.
A kind of method that ddPCR detects C.perfringens and cpa genes, it is characterised in that include the following steps：
A, sample DNA is extracted；
B, above-mentioned 20.0 μ l reaction systems are added in each reactive component, 70.0 μ l mineral oil is then added, are shifted after mixing
Droplet is automatically generated on to drop generator；
C, the droplet of generation is carefully transferred completely into 96 hole reaction plate PCR reaction tubes；Again by 96 hole reaction plates in sealer
Instrument upper sealing film is placed in regular-PCR instrument and carries out PCR reactions.
D, droplet fluorescence detector application software is opened, the 96 hole reaction plates of PCR after reaction are inserted directly into equipment,
The PCR response situations of droplet, finally calculate the copy number of testing gene according to amber pine distribution law in the every PCR reaction tubes of detection.
The method of detection C.perfringens and cpa as described above, it is characterised in that the reaction interval of PCR amplification in step B
Sequence carries out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 58.6 DEG C of annealing 1min carry out 40 cycles altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
The method of detection C.perfringens and cpa genes as described above, it is characterised in that extract sample described in step A
DNA is as follows：
Using rotary centrifugal column for nucleic acid partition method, the extraction of DNA of bacteria suitable for pure bacterium solution and secretion.Specifically
Operation：Picking is added in 200 μ l sterilizing redistilled waters through suspicious single bacterium colony, prepares C.perfringens bacteria suspension, set
In 100 DEG C of heating 20min on dry-type thermostat, 10min then is centrifuged in 12000r/min, supernatant is taken to dispense Eppendorf
Pipe, the crude templates of DNA as amplified reaction are dissolved in the TE solution of 50 μ L.
Sensibility 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 carries out
When Blast compares, sequence with Genbank aim sequence very high homologies.10 times of diluted reference strain genomic DNAs are added
Previous reaction system repeats have repeatability very well in experiment display detection sample.It dilutes template concentrations logarithm and copies
Also it is in good linear relationship R between shellfish numerical value2≥0.95.Illustrate that this method has preferable accuracy and good stabilization
Property.
Compared with prior art, the present invention haing the following advantages:
1) reagent constituents proportioning of the present invention is reasonable, and easy to use, detection is quick, accurately；
2) detection method simplifies testing process, and is not necessarily to make standard curve, shortens detection cycle, and
Without anaerobic device；
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
To be copied down to 1, therefore, detection accurate and that repeatability is good is carried out to the slight change of low concentration mrna concentration.
5) it can be realized in same reaction system using detection method while C.perfringens and cpa carry out essence
Quasi- detection.With preferable industrialization prospect.
Specific implementation mode
The present invention is described further With reference to embodiment：
Embodiment 1
The present invention detects the primer of C.perfringens and cpa in food, and the sequence of the primer is respectively：
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
Embodiment 2
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
Embodiment 3
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The content ratio of the sense primer recAF and sense primer cpaF is 0.1:1.The downstream primer
The content ratio of recAR and downstream primer cpaF is 0.1:1.
Embodiment 4
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The content ratio of the sense primer recAF and sense primer cpaF is 0.5:1.The downstream primer
The content ratio of recAR and downstream primer cpaF is 0.5:1.
Embodiment 5
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The content ratio of the sense primer recAF and sense primer cpaF is 0.5:1.The downstream primer
The content ratio of recAR and downstream primer cpaF is 0.1:1.
Embodiment 6
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The content ratio of the sense primer recAF and sense primer cpaF is 0.1:1.The downstream primer
The content ratio of recAR and downstream primer cpaF is 0.5:1.
Embodiment 3
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The content ratio of the sense primer recAF and sense primer cpaF is 1:1.The downstream primer recAR
Content ratio with downstream primer cpaF is 1:1.
Embodiment 8
The kit of C.perfringens and cpa in a kind of detection food of the present invention, wherein 20 μ L reaction systems include with
Lower component：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The content ratio of the sense primer recAF and sense primer cpaF is 0.1:1.The downstream primer
The content ratio of recAR and downstream primer cpaF is 1:1.
Embodiment 9
The present invention detects the method for C.perfringens and cpa in food, includes the following steps：
A, sample DNA is extracted；
B, the 20.0 μ l reaction systems as described in embodiment 2-8 is any are added in each reactive component, 70.0 μ l is then added
Mineral oil is transferred on drop generator after mixing and automatically generates droplet；
C, the droplet of generation 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 is placed in regular-PCR instrument and carries out PCR reactions.
D, droplet fluorescence detector application software is opened, the 96 hole reaction plates of PCR after reaction are inserted directly into equipment,
The PCR response situations of droplet, finally calculate the copy number of testing gene according to amber pine distribution law in the every PCR reaction tubes of detection.
Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
The fluorescent PCR amplification carries out according to the following steps：
(1) (1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 58.6 DEG C of annealing 1min carry out 40 cycles altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
Embodiment 10
Picking is added in 200 μ l sterilizing redistilled waters through suspicious single bacterium colony, prepares C.perfringens bacteria suspension, will
It is placed in 100 DEG C of heating 20min on dry-type thermostat, then centrifuges 10min in 12000r/min, supernatant is taken to dispense
Eppendorf is managed, and the crude templates of DNA as amplified reaction are dissolved in the TE solution of 50 μ L.Then 4.0 μ L extracts are taken
It is added to following reaction system：
10.0 μ L of wherein 2 × ddPCR Super Mix, C.perfringens and each 1.0 μ L of the upstream and downstream cpa primer, it visits
Each 1.0 μ L of needle, 4.0 μ L of DNA profiling.Wherein primer sequence is as follows:
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer NetBF：acggaaaaatgaaatggcctga
Downstream primer NetBR：tcgccattgagtagtttccca
Probe NetBP：aggaaaaactgctggtgctggaa.
PCR amplification is carried out, is carried out according to the following steps：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 58.6 DEG C of annealing 1min carry out 40 cycles 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, carrying out C.perfringens quantity survey
Fixed control.In triplicate, results are averaged for C.perfringens for experiment.The results show that the present invention uses digital pcr method
To the measurement result of C.perfringens quantity in food, the total plate count results relevance detected with classical culture protocols is high
Up to 98.96%, this illustrates that this method has very high accuracy.Single sample whole process inspection of method in addition, digital pcr of the present invention is enjoyed oneself to the full
It is 4 hours to survey the time, considerably shorter than existing traditional tablet culture method of counting, and detection C.perfringens is simultaneously, moreover it is possible to cpa
Gene carries out accurate quantitative analysis detection, is capable of the propagation risk of accurate Fast Evaluation food-borne pathogens.Therefore there is good skill
Art advantage and Developmental Prospect of Industrialization.
The basic principles and main features and advantages of the present invention of the present invention have been shown and described above.The skill of the industry
Art personnel it should be appreciated that the present invention is not limited to the above embodiments, the above embodiments and description only describe
The principle of the present invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, 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.
SEQUENCE LISTING
<110>Inspection and Quarantine Center of Zhongshan Entry-Exit Inspection and Quarantine Bureau
<120>Detect the primer of C.perfringens and plc genes, kit and method in food
<130>Detect the primer of C.perfringens and plc genes, kit and method in food
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Clostridium perfringens
<400> 1
tgggagattc tcacgttggt c 21
<210> 2
<211> 21
<212> DNA
<213> Clostridium perfringens
<400> 2
gcttgcttaa atggaggagc a 21
<210> 3
<211> 22
<212> DNA
<213> Clostridium perfringens
<400> 3
acaacaccag gtggtagagc gt 22
<210> 4
<211> 20
<212> DNA
<213> Clostridium perfringens
<400> 4
cttatttgtg ccgcgctagc 20
<210> 5
<211> 20
<212> DNA
<213> Clostridium perfringens
<400> 5
gtcctgcgct atcaacagca 20
<210> 6
<211> 25
<212> DNA
<213> Clostridium perfringens
<400> 6
tgggctggga catcaactaa agtct 25
Claims (7)
1. the primer of C.perfringens and cpa genes in a kind of detection food, it is characterised in that primer and probe combination
Sequence is respectively：
Sense primer recAF:tgggagattctcacgttggtc
Downstream primer recAR:gcttgcttaaatggaggagca
Probe recAP:acaacaccaggtggtagagcgt
Sense primer cpaF：tccaaagcctttaaatgagaagga
Downstream primer cpaR：cccgctatttccctctgtgt
Probe cpaP：ggcaaaaagagtactagttgagcgga.
2. the kit of C.perfringens and cpa genes in a kind of detection food, it is characterised in that 20.0 μ L in the kit
Reaction system includes following components：2 × ddPCR Super Mix, 10.0 μ L, each 1.0 μ L of upstream and downstream primer, each 1.0 μ of probe
L, 4.0 μ L of DNA profiling.
3. kit according to claim 2, it is characterised in that the sense primer recAF and sense primer cpaF's
Content ratio is 0.1-1:1.
4. kit according to claim 2 or 3, it is characterised in that the downstream primer recAR and downstream primer
The content ratio of cpaR is 0.1-1:1.
5. a kind of method of C.perfringens and cpa genes in detection food, it is characterised in that include the following steps：
A, sample DNA is extracted；
B, 20.0 μ l reaction systems as claimed in claim 2 are added in each reactive component, 70.0 μ l mineral oil is then added, mixed
It is transferred on drop generator after even and automatically generates droplet；Take the positive quality control in the kit and negative matter respectively simultaneously
Control handles according to method identical with step A, 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；96 hole reaction plates are placed in regular-PCR instrument and carry out PCR reactions in sealer instrument upper sealing film again；
D, droplet fluorescence detector application software is opened, the 96 hole reaction plates of PCR after reaction are inserted directly into equipment, is detected
The PCR response situations of droplet, finally calculate the copy number of testing gene according to amber pine distribution law in per PCR reaction tubes.
6. detection method according to claim 5, it is characterised in that the response procedures of PCR amplification press following step in step C
It is rapid to carry out：
(1) 94 DEG C of pre-degeneration 3min；
(2) 94 DEG C of denaturation 10s, 58.6 DEG C of annealing 1min carry out 40 cycles altogether；
(3) 98 DEG C of enzyme heat inactivation 10min；
(4) 4 DEG C stop reaction.
7. detection method according to claim 5, it is characterised in that the extraction sample DNA described in step A：Picking warp can
Doubtful single bacterium colony is added in 200 μ l sterilizing redistilled waters, prepares C.perfringens bacteria suspension, place it in dry-type thermostat
Upper 100 DEG C of heating 20min, then centrifuges 10min in 12000r/min, takes supernatant packing Eppendorf pipes, anti-as amplification
The crude templates of DNA answered, are dissolved in the TE solution of 50 μ L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810176258.5A CN108441568A (en) | 2018-03-02 | 2018-03-02 | Detect the primer of C.perfringens and cpa genes, kit and method in food |
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| CN201810176258.5A CN108441568A (en) | 2018-03-02 | 2018-03-02 | Detect the primer of C.perfringens and cpa genes, kit and method in food |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531867A (en) * | 2014-12-25 | 2015-04-22 | 湖北省农业科学院畜牧兽医研究所 | Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit |
| CN107513561A (en) * | 2017-07-28 | 2017-12-26 | 中山出入境检验检疫局检验检疫技术中心 | Detect primer, kit and the method for P. aeruginosa and C.perfringens |
-
2018
- 2018-03-02 CN CN201810176258.5A patent/CN108441568A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531867A (en) * | 2014-12-25 | 2015-04-22 | 湖北省农业科学院畜牧兽医研究所 | Clostridium perfringens enterotoxin positive bacteria dual fluorescent quantitative PCR rapid detection kit |
| CN107513561A (en) * | 2017-07-28 | 2017-12-26 | 中山出入境检验检疫局检验检疫技术中心 | Detect primer, kit and the method for P. aeruginosa and C.perfringens |
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| Title |
|---|
| B. HELEN JOST等: "Clonal relationships among Clostridium perfringens of porcine origin as determined by multilocus sequence typing", 《VETERINARY MICROBIOLOG》 * |
| 周明东: "食源性致病菌快速检测方法的建立及其试剂盒的研制与应用", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 康文娟: "鸡源产气荚膜梭菌多重PCR鉴定和药敏实验", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
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