CN101875967B - Method for quickly testing food-borne pathogenic bacteria - Google Patents
Method for quickly testing food-borne pathogenic bacteria Download PDFInfo
- Publication number
- CN101875967B CN101875967B CN2009102450327A CN200910245032A CN101875967B CN 101875967 B CN101875967 B CN 101875967B CN 2009102450327 A CN2009102450327 A CN 2009102450327A CN 200910245032 A CN200910245032 A CN 200910245032A CN 101875967 B CN101875967 B CN 101875967B
- Authority
- CN
- China
- Prior art keywords
- food
- sequencing
- reaction
- primer
- tetra
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for quickly testing food-borne pathogenic bacteria. In the method, genomic DNAs of salmonella, staphylococcus aureus, Shigella and Listeriosis are taken as templates, genbank data retrieval tools BLAST and ICB respectively perform homology analysis on the salmonella, staphylococcus aureus, Shigella and Listeriosis, and the four food-borne pathogenic bacteria are tested by combining pyrosequencing technology. The test method has the characteristics of high efficiency, quickness, accuracy, simple and convenient operation and the like, and opens up wide prospect for quickly testing the food-borne pathogenic bacteria.
Description
Technical field
The invention belongs to food microorganisms identifies and applied technical field; Be specifically related to the authentication method of Salmonellas, streptococcus aureus, Shigellae and four kinds of food-borne pathogens of listeria spp, particularly a kind of tetra-sodium sequencing technologies that utilizes is to four kinds of food-borne pathogens rapid identification method.
Background technology
Estimate that according to the World Health Organization whole world has among hundreds of millions of food origin disease patients every year, 70% pathogeny is various invasive organisms.Nutrition and Food Safety Office of China Disease Prevention and control Centre has carried out the successive active monitoring to the pathogenic bacterium pollution situation in raw meat, cold cuts, breast and the milk-product of China part provinces and cities, fishery products, the vegetables in recent years; The result shows; Mikrobe property food poisoning ranks first; Account for 39.62%, chemical food poisoming accounts for 38.56%, and of animal or plant nature and agnogenic food poisoning are all about 10%.
China in 2003 detects national standard to pathogenic bacterium (Salmonellas, listeria bacteria, streptococcus aureus, Bacillus cereus etc.) again and revises; But compare too big different with national standard in 1994; Be main with traditional separation and Culture detection method still, this method is except that complex operation, and proving time is longer; Generally need cultivation in 5～7 days, biochemical identification time, can not satisfy the modern requirement that detects far away.The external detection to pathogenic microorganism mainly adopted immunological method and color developing culture medium method and mikrobe automatic identifying system at present, and aforesaid method common advantage is exactly to save certain qualification time, but shortcoming is also very obvious.Wherein the shortcoming of immunological method is: the requirement of operative technique property is high, and false negative is high, popularizes difficulty; The shortcoming of color developing culture medium is unstable, and period of storage is short, influenced greatly by extraneous factor; The mikrobe automatic identifying system, China also fails to produce full automatic Bacteria Identification plant and instrument at present, and the also complete dependence on import of matched reagent.
Along with the develop rapidly of life science, what various molecular biology experiments were technological continues to bring out, and expands to the Micro biological Tests field gradually.Various is that basic molecular biology inspection technology is widely used in microorganism detection in recent years with the round pcr; But these technology only can provide the clip size information of positive amplified production; And can't show specific gene order; False negative and false positive can't be avoided, and therefore are not enough to provide the conclusive evidence conclusion.
Tetra-sodium order-checking (Pyrosequencing) technology is a dna sequence analysis technology of new generation, and this technology need not be carried out electrophoresis, and dna fragmentation also need not fluorescent mark, operates very easy.The tetra-sodium sequencing technologies is by the enzyme cascade chemiluminescence reaction in 4 kinds of enzymatic same reaction systems; Take turns in the sequencing reaction at each; Only add a kind of dNTP; If this dNTP and template are matched, polysaccharase just can be incorporated into it in primer strand and the tetra-sodium group (PPi) of mole number such as discharges.PPi can finally be converted into visible light signal, and is converted into a peak value by PyrogramTM.The Nucleotide number that mixes is directly proportional in the height of each peak value and the reaction.Add a kind of dNTP down then, continue the synthetic of DNA chain,, reach the purpose of The real time measure dna sequence dna through detecting the release and the intensity of fluorescence.This technology is used to measure the short dna chain-ordering; Be present unique technology that can obtain quantitative sequence results in real time, have the susceptibility of round pcr and the accuracy of sequencing technologies concurrently, accuracy is high; Good reproducibility; Level of automation is high, and is simple to operate, is suitable for very much food-borne pathogens and identifies fast and accurately.Up to now, the tetra-sodium sequencing technologies that utilizes of Shang Weiyou success is identified the bibliographical information of Salmonellas, streptococcus aureus, Shigellae and four kinds of food-borne pathogens of listeria spp.
Summary of the invention
The objective of the invention is to disclose a kind of food-borne pathogens rapid identification method and, judged four kinds of food-borne pathogens through the tetra-sodium sequencing result according to the conserved sequence of four kinds of pathogenic bacterium 16sRNA design PCR specificity amplification primer and sequencing primer.Detection method of the present invention has high efficiency, fast, precisely reaches characteristics such as easy and simple to handle.For realizing above-mentioned purpose, the present invention provides following technical scheme:
Be used to identify the pcr amplification and the tetra-sodium Auele Specific Primer of four kinds of food-borne pathogens; It is characterized in that; Comprise primer forward sequence: 5 '-AGCAGCCGCGGTAATACG-3 '; Reverse primer sequence: 5 '-Biotin-CGCATTTCACCGCTACAC-3 ', sequencing primer sequence: 5 '-ATTTATTGGGCGTAAAG-3 '.Every primer is mixed with the stock solution that concentration is 100 μ mol/L respectively, and working concentration is 10 μ mol/L.
The present invention adopts the method for pcr amplification and four kinds of food-borne pathogens of tetra-sodium Auele Specific Primer Rapid identification, it is characterized in that as follows:
(1) adopts positive and negative PCR special primer and
Master Mix instant 2X reaction buffer, add four kinds of food-borne pathogens template DNAs and carry out pcr amplification;
Pcr amplification reaction system wherein: the TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mix instant 2X reaction buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers; Template DNA 2 μ L are with sterilization deionized water polishing to 50 μ L; Response procedures: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations;
(2) adopt the order-checking Auele Specific Primer, the pcr amplification product of four kinds of food-borne pathogens is carried out the tetra-sodium order-checking;
Tetra-sodium sequencing reaction system wherein: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L, Sepharose Beads 3 μ L; Binding Buffer 47 μ L (10mmol/L Tris-HCl, 2mol/L NaCl, 1mmol/L EDTA; 0.1%Tween20, pH7.6), 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L (20mmol/LTris-AC, 2mmol/L MgAc2, pH7.69).
(3) behind the employing tetra-sodium sequenator order-checking 18min, directly be judged to be which kind of food-borne pathogens respectively through visible light signal peak value and four kinds of food-borne pathogens known arrays contrast to be determined.
Rapid identification method of the present invention, wherein four kinds of food-borne pathogens refer to: Salmonellas, streptococcus aureus, Shigellae and listeria spp.
Template DNA refers to and supplies test agent bacterial genomes DNA in the detection method of the present invention.
Tetra-sodium order-checking of the present invention is the enzyme cascade chemiluminescence reaction in archaeal dna polymerase, ATP sulfurylase, luciferase and 4 kinds of enzymatic same reaction systems of apyrase; Take turns in the sequencing reaction at each; Only add a kind of dNTP; If this dNTP and template are matched, polysaccharase just can be incorporated into it in primer strand and the tetra-sodium group (PPi) of mole number such as discharges.PPi can finally be converted into visible light signal, and is converted into a peak value by PyrogramTM.The Nucleotide number that mixes is directly proportional in the height of each peak value and the reaction.Add a kind of dNTP down then, continue the synthetic of DNA chain,, reach the purpose of The real time measure dna sequence dna through detecting the release and the intensity of fluorescence.
For measuring method of the present invention can more clearly be described, do with detailed explanation in the face of TP of the present invention down.
1, principle
Present method is used a kind of its principle of novel nucleic acid sequence measurement: under the synergy of archaeal dna polymerase, ATP sulfurylase, luciferase and 4 kinds of enzymes of apyrase; Each dNTP polymerization and first order fluorescence signal on the primer are discharged coupling to get up; Through detecting the release and the intensity of fluorescence, reach the purpose of The real time measure dna sequence dna.Has the accuracy height, good reproducibility, level of automation height and simple operation and other advantages.
2, design of primers
The present invention is according to conserved sequence 2 pcr amplification Auele Specific Primers of design and 1 sequencing primer of four kinds of pathogenic bacterium 16sRNA.Primer is synthetic by Shanghai bio-engineering corporation.
Table 1 primer sequence table is following:
3, reaction conditions
Pcr amplification reaction reagent needs
Master Mix instant 2X reaction buffer, pcr amplification primer and template DNA.Sequencing reaction reagent needs pcr amplification product, SepharoseBeads, Binding Buffer, sequencing primer and Annealing Buffer, needs the tetra-sodium sequenator, order-checking time 18min.
Materials and methods:
(1) reagent:
Master Mixes solution that Promega company produces; Worker's synthetic specificity amplification primer and sequencing primer are given birth in Shanghai; The SepharoseBead that Biotage company produces.
(2) amplification reaction system and amplification program: the TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mix instant 2X reaction buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers; Template DNA 2 μ L are with sterilization deionized water polishing to 50 μ L; Response procedures: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations.
(3) sequencing reaction system: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L, Sepharose Beads 3 μ L; Binding Buffer 47 μ L (10mmol/L Tris-HCl, 2mol/L NaCl, 1mmol/L EDTA; 0.1%Tween20, pH7.6), 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L (20mmol/LTris-AC, 2mmol/L MgAc
2, pH7.6);
(4) after the order-checking of employing tetra-sodium sequenator, directly be judged to be which kind of food-borne pathogens respectively through visible light signal peak value and four kinds of food-borne pathogens known arrays contrast to be determined, the order-checking time is 18min.
The present invention adopts rapid identification method that pcr amplification and tetra-sodium Auele Specific Primer be used for food-borne pathogens compared with prior art, has the following advantages:
(1) easy and simple to handle: programming operations does not need the particular theory basis.
(2) accuracy: this invention is to be the basis with the tetra-sodium sequencing technologies, has very high accuracy.
(3) rapidly and efficiently: can accomplish food-borne pathogenic dientification of bacteria work within the 3h;
(4) result judges simply: qualification result can directly be read from the visible light signal peak value.
Description of drawings:
Fig. 1 is the electrophoretic analysis collection of illustrative plates of amplified production.Be followed successively by Marker, Salmonellas 1, Salmonellas 2, Salmonellas 3, streptococcus aureus 1, streptococcus aureus 2, streptococcus aureus 3, Shigellae 1, Shigellae 2, Shigellae 3, listeria spp 1, listeria spp 2, listeria spp 3 from left to right.
Fig. 2 is Salmonellas amplified production sequencing result figure.
Fig. 3 is streptococcus aureus amplified production sequencing result figure.
Fig. 4 is Shigellae amplified production sequencing result figure.
Fig. 5 is listeria spp amplified production sequencing result figure.
Embodiment
For method of the present invention can more clearly be described; Do with detailed explanation in the face of TP of the present invention down; Wherein archaeal dna polymerase, ATP sulfurylase, luciferase and 4 kinds of enzymes of apyrase all have commercially availablely, and what need in addition to explain is: 1, primer sequence of the present invention is seen table 1.2, a small amount of preparation method (seeing Appendix) of the extraction of four kinds of food-borne pathogens template DNAs: bacterial genomes DNA.
Embodiment 1
(1) reagent:
Master Mix instant 2X reaction buffer that Promega company produces; Worker's synthetic specificity amplification primer and sequencing primer are given birth in Shanghai; The Sepha rose Bead that Biotage company produces.
(2) amplification reaction system: the TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mix instant 2X reaction buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers; Salmonellas bacterial genomes template DNA 2 μ L are with sterilization deionized water polishing to 50 μ L;
The amplified reaction program: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations.
(3) sequencing reaction system: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L, Sepharose Beads 3 μ L; Binding Buffer 47 μ L (10mmol/L Tris-HCl, 2mol/L NaCl, 1mmol/L EDTA; 0.1%Tween20, pH7.6), 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L (20mmol/LTris-AC, 2mmol/L MgAc
2, pH7.6), opposite position adds required substrate, enzyme and dNTP in the reagent cabin according to software prompt.
(4) adopt the tetra-sodium sequenator; Check order according to the software prompt editor; After order-checking finishes, directly be judged to be which kind of food-borne pathogens respectively through visible light signal peak value and four kinds of food-borne pathogens known arrays contrast to be determined, the order-checking time is 18min.The result is as shown in Figure 2, and sequencing result can directly read as CGCACGCAGGCGGTCT through the visible light signal peak value, and supplying the test agent decidable is Salmonellas.
Embodiment 2
(1) reagent:
Master Mix instant 2X reaction buffer that Promega company produces; Worker's synthetic specificity amplification primer and sequencing primer are given birth in Shanghai; The Sepharose Bead that Biotage company produces.
(2) amplification reaction system and amplification program: the TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mix instant 2X reaction buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers; Streptococcus aureus bacterial genomes template DNA 2 μ L are with sterilization deionized water polishing to 50 μ L;
The amplified reaction program: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations.
(3) sequencing reaction system: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L, Sepharose Beads 3 μ L; Binding Buffer 47 μ L (10mmol/L Tris-HCl, 2mol/L NaCl, 1mmol/L EDTA; 0.1%Tween20, pH7.6), 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L (20mmol/LTris-AC, 2mmol/L MgAc
2, pH7.6); Opposite position adds required substrate, enzyme and dNTP in the reagent cabin according to software prompt.
(4) adopt the tetra-sodium sequenator; Check order according to the software prompt editor; After order-checking finishes, directly be judged to be which kind of food-borne pathogens respectively through visible light signal peak value and four kinds of food-borne pathogens known arrays contrast to be determined, the order-checking time is 18min.The result is as shown in Figure 3, and sequencing result can directly read as CGCGCGTAGGCGGTTT through the visible light signal peak value, and supplying the test agent decidable is streptococcus aureus.
Embodiment 3
(1) reagent:
Master Mix instant 2X reaction buffer that Promega company produces; Worker's synthetic specificity amplification primer and sequencing primer are given birth in Shanghai; The Sepharose Bead that Biotage company produces.
(2) amplification reaction system and amplification program: the TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mix instant 2X reaction buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers; Shigellae bacterial genomes template DNA 2 μ L are with sterilization deionized water polishing to 50 μ L;
The amplified reaction program: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations.
(3) sequencing reaction system: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L, Sepharose Beads 3 μ L; Binding Buffer 47 μ L (10mmol/L Tris-HCl, 2mol/L NaCl, 1mmol/L EDTA; 0.1%Tween20, pH7.6), 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L (20mmol/LTris-AC, 2mmol/L MgAc
2, pH7.6); Opposite position adds required substrate, enzyme and dNTP in the reagent cabin according to software prompt.
(4) adopt the tetra-sodium sequenator; Check order according to the software prompt editor; After order-checking finishes, directly be judged to be which kind of food-borne pathogens respectively through visible light signal peak value and four kinds of food-borne pathogens known arrays contrast to be determined, the order-checking time is 18min.The result is as shown in Figure 4, and sequencing result can directly read as CGCACGCAGGCGGTTT through the visible light signal peak value, and supplying the test agent decidable is Shigellae.
Embodiment 4
(1) reagent:
Master Mix instant 2X reaction buffer that Promega company produces; Worker's synthetic specificity amplification primer and sequencing primer are given birth in Shanghai; The Sepharose Bead that Biotage company produces.
(2) amplification reaction system and amplification program: the TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mix instant 2X reaction buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers; Monokaryon hyperplasia listeria spp bacterial genomes template DNA 2 μ L are with sterilization deionized water polishing to 50 μ L;
The amplified reaction program: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations.
(3) sequencing reaction system: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L, Sepharose Beads 3 μ L; Binding Buffer 47 μ L (10mmol/L Tris-HCl, 2mol/L Na Cl, 1mmol/L EDTA; 0.1%Tween20, pH7.6), 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L (20mmol/LTris-AC, 2mmol/L MgAc
2, pH7.6); Opposite position adds required substrate, enzyme and dNTP in the reagent cabin according to software prompt.
(4) adopt the tetra-sodium sequenator; Check order according to the software prompt editor; After order-checking finishes, directly be judged to be which kind of food-borne pathogens respectively through visible light signal peak value and four kinds of food-borne pathogens known arrays contrast to be determined, the order-checking time is 18min.The result is as shown in Figure 5, and sequencing result can directly read as CGCGCGCAGGCGGTCT through the visible light signal peak value, and supplying the test agent decidable is monokaryon hyperplasia listeria spp.
Embodiment 5 (contrast experiment)
(1) sample adds: with the Salmonellas is example; With 10 times of gradient dilutions of Salmonellas bacterium liquid; Filter out the bacteria suspension of 4 * 104CFU/mL, 4 * 103CFU/mL, 4 * 102CFU/mL, 4 * 101CFU/mL, 4CFU/mL, 0CFU/mL, respectively get 1mL and make an addition in the 25mL milk, subsequent use.
(2) increase bacterium before carrying out according to traditional national standard method: above-mentioned 25mL milk is added in the 225mL sterilization BPW solution, cultivate 18h for 37 ℃.
(3) traditional National Standard Method and tetra-sodium sequence measurement compare experiment:
1. traditional national standard method: above-mentioned BPW nutrient solution is respectively got 1mL be connected to respectively among 10mLTTB and the SC; Continue to cultivate; It is dull and stereotyped to be inoculated in BS and HE selectivity after the cultivation; Carry out biochemical identification after waiting to grow colonies typical, qualification result is that the interpolation sample of 4 * 104CFU/mL, 4 * 103CFU/mL, 4 * 102CFU/mL, 4 * 101CFU/mL, 4CFU/mL all detects, and the interpolation sample of 0CFU/mL does not detect.
2. tetra-sodium sequence measurement: a small amount of preparation method of above-mentioned BPW nutrient solution respectively being got 2mL bacterial genomes DNA extracts template DNA; Be used for pcr amplification; The TV of amplified reaction is 50 μ L; Its various compositions are respectively:
Master Mixes Buffer 25 μ L; Each 1 μ L of 10 μ mol/L primers supplies test agent template DNA 2 μ L, with sterilization deionized water polishing to 50 μ L; The amplified reaction program: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30S, 53 ℃ of annealing 30S, 72 ℃ are extended 30S, circulate 30 times, and last 72 ℃ are extended 5min, 4 ℃ of preservations.Then carry out the tetra-sodium sequencing reaction, the sequencing reaction system: the TV of sequencing reaction is 100 μ L, and wherein various compositions are respectively: PCR product 50 μ L; Sepharose Beads 3 μ L, Binding Buffer 47 μ L (10mmol/LTris-HCl, 2mol/L NaCl; 1mmol/L EDTA, 0.1%Tween20, pH7.6); 10 μ mol/L sequencing primers, 1.2 μ L; Annealing Buffer 38.8 μ L, opposite position adds required substrate, enzyme and dNTP in the reagent cabin according to software prompt.Sequencing result all detects with the interpolation sample that is 4 * 104CFU/mL, 4 * 103CFU/mL, 4 * 102CFU/mL, 4 * 101CFU/mL, 4CFU/mL, and the interpolation sample of 0CFU/mL does not detect.
(4) contrast and experiment: traditional National Standard Method is consistent with tetra-sodium sequence measurement detected result.Annex: the template DNA that supplies a small amount of preparation method extraction Salmonellas, streptococcus aureus, Shigellae and the listeria spp of test agent bacterial genomes DNA:
(1) gets the 2mL bacterial cultures, the centrifugal 2min of 12000r/min;
(2) throw out adds the TE damping fluid of 567 μ L, vortex mixing.Add the SDS of 30 μ L10% and the Proteinase K of 3 μ L20mg/mL, put upside down mixing, in 37 ℃ of incubation 1h;
(3) add 100 μ L5mol/LnaCl, fully mixing adds 80 μ LCTAB/NaCl solution again, and mixing is in 65 ℃ of incubation 10min;
(4) add isopyknic chloroform/primary isoamyl alcohol, put upside down mixing, centrifugal 5min.Supernatant is changed in the new pipe;
(5) add isopyknic phenol/chloroform/primary isoamyl alcohol, put upside down mixing, centrifugal 5min changes supernatant in the new pipe over to;
(6) add 0.6 times of volume Virahol, put upside down mixing gently, place 30min for-20 ℃, the centrifugal 5min of 12000r/min abandons supernatant;
(7) 500 μ L70% washing with alcohol deposition, the centrifugal 5min of 12000r/min abandons supernatant, treats that deposition is dry, heavily is dissolved in the 100 μ L deionized waters, is the bacterial gene template DNA that supplies test agent.
Sequence table
< 110>Centralab Tianjin Academy of Agriculture Science
< 120>a kind of rapid identification method of food-borne pathogens
<160>3
<210>1
<211>18bp
<212>DNA
< 213>artificial sequence
<400>1
agcagccgcg?gtaatacg 18
<210>2
<211>18bp
<212>DNA
< 213>artificial sequence
<400>2
cgcatttcac?cgctacac 18
<210>3
<211>17bp
<212>DNA
< 213>artificial sequence
<400>3
atttattggg?cgtaaag 17
Claims (1)
1. be used to identify the pcr amplification and the tetra-sodium Auele Specific Primer of four kinds of food-borne pathogens; It is characterized in that; Comprise primer forward sequence: 5 '-AGCAGCCGCGGTAATACG-3 '; Reverse primer sequence: 5 '-Biotin-CGCATTTCACCGCTACAC-3 ', sequencing primer sequence: 5 '-ATTTATTGGGCGTAAAG-3 '; Wherein said four kinds of food-borne pathogens refer to: Salmonellas, streptococcus aureus, Shigellae and listeria spp.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102450327A CN101875967B (en) | 2009-12-23 | 2009-12-23 | Method for quickly testing food-borne pathogenic bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102450327A CN101875967B (en) | 2009-12-23 | 2009-12-23 | Method for quickly testing food-borne pathogenic bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101875967A CN101875967A (en) | 2010-11-03 |
| CN101875967B true CN101875967B (en) | 2012-05-30 |
Family
ID=43018646
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102450327A Expired - Fee Related CN101875967B (en) | 2009-12-23 | 2009-12-23 | Method for quickly testing food-borne pathogenic bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101875967B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816844B (en) * | 2012-08-08 | 2014-07-02 | 许龙岩 | Primer, kit and method for detecting staphylococcus aureus through PCR(Polymerase Chain Reaction)-pyrophosphate method |
| CN102827931B (en) * | 2012-08-15 | 2014-05-14 | 广东出入境检验检疫局检验检疫技术中心 | Detection primer for detecting listeria monocytogenes by PCR-pyrophosphate method, kit and detection method |
| CN106636452A (en) * | 2017-03-08 | 2017-05-10 | 海南出入境检验检疫局热带植物隔离检疫中心 | Rapid identification method of imported plant quarantine pest saperda octopunctata scopoli |
| CN107338294B (en) * | 2017-07-17 | 2018-06-19 | 贵州省产品质量监督检验院 | A kind of quadruple real time fluorescent PCR method for detecting 4 kinds of pathogenic bacteria |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285090A (en) * | 2008-05-21 | 2008-10-15 | 厦门市疾病预防控制中心 | Detection process for food-borne pathogenic bacteria |
-
2009
- 2009-12-23 CN CN2009102450327A patent/CN101875967B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285090A (en) * | 2008-05-21 | 2008-10-15 | 厦门市疾病预防控制中心 | Detection process for food-borne pathogenic bacteria |
Non-Patent Citations (2)
| Title |
|---|
| 孟成艳等.PCR一焦磷酸测序法快速检测和鉴定临床常见致病菌的研究.《中华检验医学杂志》.2007,第30卷(第4期),第456-458页. * |
| 杨国兴.多重PCR检测肉及肉制品中四种食源性致病菌的研究.《中国优秀硕士学位论文全文数据库 工程科技I辑》.2008,(第08期),B024-45. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101875967A (en) | 2010-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111073989B (en) | Rapid constant-temperature detection method and application of shigella nucleic acid | |
| CN107109402B (en) | Compositions for reducing inhibition of nucleic acid amplification | |
| CA2506574A1 (en) | One step real-time rt pcr kits for the universal detection of organisms in industrial products | |
| CN103320434A (en) | Salmonella LAMP (loop-mediated isothermal amplification) primer group and kit and detection method | |
| Liu et al. | Viable but nonculturable (VBNC) state, an underestimated and controversial microbial survival strategy | |
| CN106967816A (en) | Detect the RPA specific primers of vibrio parahaemolytious and kit and application in food | |
| CN101875967B (en) | Method for quickly testing food-borne pathogenic bacteria | |
| CN102154497B (en) | M-PCR (Multiplex Polymerase Chain Reaction) primers, probes and detection methods for vibrio cholerae, vibrio parahaemolyticus and salmonella | |
| US20220098645A1 (en) | Fast and portable microfluidic detection system as an alternative to salmonella's classical culture method | |
| CN102676664A (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| CN102719542A (en) | Simultaneous amplification and testing reagent kit for VC (vibrio cholerae) | |
| CN103443294B (en) | For the method for modification of nucleic acids | |
| CN103451310B (en) | Gene chip capable of simultaneously detecting various vibrios and method for detecting vibrios | |
| CN103436623A (en) | Rapid detection kit for viable salmonella in food and use method thereof | |
| CN104450930A (en) | Molecular detection method of vibrio parahaemolyticus and application thereof | |
| CN110564822B (en) | LAMP technology-based transgenic corn Bt 176-related gene detection method and kit | |
| CN101805788B (en) | PCR specific amplified primer for fast detection of vibrio parahaemolyticus based on SCAR molecular markers and detection kit | |
| CN108220460B (en) | Food-borne streptococcus pyogenes LAMP primer group, kit and application | |
| KR101846182B1 (en) | Primer sets for simultaneous detection of Staphylococcus aureus, Bacillus cereus and Salmonella spp., polymerase chain reaction kit thereof | |
| CN114164296B (en) | Primer probe composition for detecting pythium oligandrum, kit and application and detection method | |
| CN105695599A (en) | Molecular beacon probe capable of rapidly detecting rifampicin drug-resistant mycobacterium tuberculosis and kit | |
| CN102382880A (en) | Bacillus pumilus RT-LAMP detection kit and detection method thereof | |
| CN106282315A (en) | The LAMP detection primer combination of a kind of Trichothecenes toxin and LAMP detection kit and LAMP method | |
| CN105331610A (en) | Quintuple PCR primers for detecting pathogenic bacteria in fresh agricultural products, probe and detection kit | |
| CN105316397A (en) | Detection primer, probe and detection method for direct fluorescence quantitative PCR (polymerase chain reaction) of salmonellae in cow milk |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120530 Termination date: 20131223 |