CN103966353A - Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method - Google Patents
Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method Download PDFInfo
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Abstract
The invention discloses a detecting method for Cronobacter sakazakii as well as a kit and primers used in the detecting method. The method comprises the following steps of (1) extracting a genome DNA (Deoxyribonucleic Acid) of a sample to be detected; (2) enabling primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2 to be subjected to PCR (Polymerase Chain Reaction) by taking the genome DNA obtained in the step (1) as a template; and (3) detecting the existence of a single amplification product 498bp in the reaction product obtained in the step (2). The primers include the primers as shown in SEQ ID NO.1 and SEQ ID NO.2. The kit comprises the primers. The method disclosed by the invention is short in detection time, low in cost, single in specificity, reliable in detecting result and simple in result judgment; and in addition, the invention provides the simple, rapid and sensitive detecting method for Cronobacter sakazakii in the technical field of food safety detection, and the detecting method has important significance for food safety in China.
Description
Technical field
The present invention relates to microorganism detection field, be specifically related to method and test kit and the primer of the rugged Crow promise of a kind of detection slope bacillus (Cronobacter sakazakii).
Background technology
The rugged Crow promise of slope bacillus (Cronobacter sakazakii) is under the jurisdiction of Crow promise Bacillaceae (Cronobacter spp.) (being former Enterobacter sakazakii), is the entozoic a kind of Gram-negative sporeless bacterium of humans and animals enteron aisle.Through clinical study, find that the rugged Crow promise of slope bacillus is modal conditioned pathogen in Crow promise Bacillaceae clinical infection case, the rugged Crow promise of slope bacillus strain can cause the infection that baby is fatal, and lethality rate is up to 40%-80%.It can cause the clinical symptom that baby is serious conventionally, for example brain purulence ulcer, meningitis, necrotizing enterocolitis and general septicemia.Once there is the report that is separated to the rugged Crow promise of slope bacillus strain in infant formula.The rugged Crow promise of slope bacillus strain is by formula powder, to endanger the essential condition pathogenic bacterium of infantile health.Ewborn infant or premature infant exist by the edible risk that has the infant formula of the rugged Crow promise of slope bacillus garrison to infect the rugged Crow promise of slope bacillus strain of polluting.In pollution section investigation, find, in the whole Infant Formula Enterprises technological process of production, the points of contamination that the rugged Crow promise of slope bacillus can be detected accounts for 31% of gross sample point.Therefore to the rugged Crow promise of slope bacillus strain Rapid identification, be, the basis of achieve effective control with differentiating.
Crow promise Bacillaceae (Cronobacter spp.) was divided into 5 novel species (wherein Cronobacter sakazakii being called to new combination), 1 the rugged Crow promise of slope bacillus gene kind (Genomospeciese) and 3 new subspecieses in 2008, to Crow promise Bacillaceae in 2012, through Multiple Sequence Alignment, find, finally determine this genus is divided into 7 kinds.Although classification and title about Crow promise bacillus become, but the Enterobacter sakazakii standard detecting method of program mode is still continued to use in current detection, with traditional biochemical reaction, identify, generally need 18～24h, and cannot divide into anyly, this obviously can not meet the demand that the rugged Crow promise of slope bacillus (Cronobacter sakazakii) is detected.Aspect discriminating, although existing amplified fragment length polymorphism (Amplified fragment length polymorphismas, AFLP), the comparison of 16S rRNA complete sequence, DNA? the multiple reliable methods such as DNA hybrid experiment and the order-checking of multidigit point, but these analytical procedures according to hereditary feature length consuming time, workload are large.Therefore, this area still lacks high efficiency evaluation and the discrimination method to the rugged Crow promise of slope bacillus (Cronobacter sakazakii) bacterial classification at present.
Summary of the invention
Technical problem to be solved by this invention is existing to defects such as the detection method length consuming time of the rugged Crow promise of slope bacillus (Cronobacter sakazakii), complex operations in order to overcome, and method and test kit and the primer of a kind of new rugged Crow promise of detection slope bacillus are provided.With method of the present invention, detect the rugged Crow promise of slope bacillus strain, detection time is short, cost is low, detected result is special, can detect specifically the rugged Crow promise of slope bacillus strain, and it is simple not judged by the interference of other kinds in Crow promise Bacillaceae (Cronobacter spp.), result, very convenient in actual applications.
One of technical scheme provided by the invention is: the method for the rugged Crow promise of the detection slope bacillus (Cronobacter sakazakii) of a kind of diagnosis of non-disease or therapeutic purpose, comprises the following steps:
(1) extract the genomic dna of testing sample;
(2) take the genomic dna of step (1) gained is template, and the primer pair with sequence as shown in SEQ ID NO.1 and SEQ ID NO.2 carries out PCR reaction; With
(3) existence of the single amplified production of 498bp in detecting step (2) gained reaction product.
According to the present invention, in step (1), the method for the genomic dna of described extraction testing sample is that this area is conventional, as uses commercially available range gene group DNA extraction test kit to operate.
In step (2), described PCR reaction is for this area routine, as long as can amplify the specific gene fragment of the rugged Crow promise of slope bacillus.
Preferably, the reaction system of described PCR reaction comprises: 1 * PCR reaction buffer, 10-15mmol/L Mg
2+, 0.2-0.3mmol/L dNTP, the sequence of 0.1-0.3 μ mol/L is the primer as shown in SEQ IDNO.1 and SEQ ID NO.2 respectively, Taq enzyme 0.05-0.1U/ μ L, and DNA profiling 10-100ng/ μ L.The response procedures of described PCR reaction is: 1. 94～95 ℃, and 3～5min; 2. 94～95 ℃, 30～40s; 3. 60～62 ℃, 30～40s; 4. 68～72 ℃, 30～40s; Step is 2. to 4. totally 30～35 circulations; 5. 68～72 ℃, 7～10min; 6. 4～15 ℃ of preservations.
More preferably, the reaction system of described PCR reaction comprises: 1 * PCR reaction buffer, 12.5mmol/LMg
2+, 0.25mmol/L dNTP, the sequence of 0.2 μ mol/L is the primer as shown in SEQ ID NO.1 and SEQ IDNO.2 respectively, Taq enzyme 0.04U/ μ L, and DNA profiling 40ng/ μ L.The response procedures of described PCR reaction is: 1. 94 ℃, and 5min; 2. 94 ℃, 30s; 3. 60 ℃, 30s; 4. 72 ℃, 30s; Step is 2. to 4. totally 35 circulations; 5. 72 ℃, 10min; 6. 12 ℃ of preservations.
In step (3), described detection can adopt the method for this area routine, is preferably detected through gel electrophoresis method, can be agarose gel electrophoresis, can be also polyacrylamide gel electrophoresis.Known according to this area routine, in the present invention, if the reaction product of step (2) gained exists single amplified production in 498bp position, illustrate and in sample to be checked, contain the rugged Crow promise of slope bacillus strain; If the reaction product of step (2) gained does not exist single amplified production in 498bp position, illustrate and in sample to be checked, do not contain the rugged Crow promise of slope bacillus strain.
Method of the present invention is particularly useful for detecting the rugged Crow promise of the slope bacillus strain in the rugged Crow promise of the slope bacillus strain, particularly milk powder in food.
Two of technical scheme provided by the invention is: the primer pair of a kind of detection slope rugged Crow promise bacillus (Cronobactersakazakii), by sequence, the primer as shown in SEQ ID NO.1 and SEQ ID NO.2 forms for it.
Three of technical scheme provided by the invention is: the test kit of a kind of detection slope rugged Crow promise bacillus (Cronobactersakazakii), it comprises aforementioned primer pair.
In the present invention, preferably, described test kit also comprises PCR damping fluid, Taq enzyme, dNTP solution and Mg
2+in one or more.More preferably, described test kit also comprises gene extraction agent and/or positive gene group DNA.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is: adopt detection method of the present invention and primer pair and test kit to detect slope rugged Crow promise bacillus (Cronobacter sakazakii) bacterial strain, detection time is short, testing cost is low, detection efficiency is high; Detection method of the present invention has single specificity, can realize specific amplification to the rugged Crow promise of slope bacillus strain, and to belonging to other interior kinds of Crow promise Bacillaceae (Cronobacter spp.) and other sibling specieses all without any amplification, detected result is reliable, result is judged simple.The present invention provides method and test kit and the primer of a kind of simple and quick sensitive rugged Crow promise of detection slope bacillus for food safety detection technical field, significant to the food safety of China.
Accompanying drawing explanation
Fig. 1 is the experimental result of PCR product after 1.5% agarose gel electrophoresis in embodiment 1.Swimming lane 1～9 is: sterilized water, Mu Tingsi Crow promise bacillus (Cronobacter muytjensii) ATCC51329, Crow promise bacillus gene kind I (Cronobacte.genomospecies I) NCTC9529, positive Crow promise bacillus (Cronobacter malonaticus) DSM18702 of malonate, Crow, Zurich promise bacillus (Cronobacter turicensis) DSM18703, Crow, Dublin promise bacillus Dublin subspecies (Cronobacter dublinensis subsp.Dublinensis) DSM18705, Crow, Dublin promise bacillus Lausanne subspecies (Cronobacter dublinensis subsp.Lausannensis) DSM18706, Crow, Dublin promise bacillus milk powder subspecies (Cronobacter dublinensis subsp.Lactaridi) DSM18707, the rugged Crow promise of slope bacillus (Cronobacter sakazakii) ATCC29544, M is DNA Marker.
Fig. 2 is the experimental result of PCR product after 1.5% agarose gel electrophoresis in embodiment 1.Swimming lane 1～32 is: the rugged Crow promise of slope bacillus (Cronobacter sakazakii ATCC29544), Salmonella typhimurium (salmonella typhimurium ATCC14023), Salmonella enteritidis (SalmonellaEnteritidis ATCC13311), enterobacter cloacae (Enterobacter cloacae ATCC13047), intestinal bacteria (Escherichia coli ATCC43889), intestinal bacteria (Escherichia coliATCC25922), Proteus mirabilis (Proteus mirabilis ATCC12453), proteus vulgaris (Proteus vulgaris ATCC33420), citrobacter (Citrobacter freundiiATCC8090), klebsiella pneumoniae (Klebsiella peneumoniae ATCC27336), klebsiella pneumoniae (Pneumonia crayresearch ATCC46114), Song Zhi Shi Shigellae (Shigellasonnei CMCC51334), shigella dysenteriae (Shigella dysenteriae CMCC51335), shigella flexneri (Shigella Flexner ATCC51371), Pseudomonas aeruginosa (Pseudomonasaeruginosa CDCB32116), Bacillus cereus (Bacillus cereous ATCC1220), smell Serratia (Serratia odorifera ATCC33077), serratia marcescens (Serratiamarcescens ATCC14040), pseudomonas putida (Pseudomonas putida ATCC17485), Pseudomonas alcaligenes (Pseudomonas alcaligenes IQCC12604), streptococcus aureus (Staphylococcus aureus ATCC29213), streptococcus aureus (Staphylococcusaureus ATCC8095), faecium (Enterococcus faecium ATCC14025), enterococcus faecalis (Enterococcus faecalis ATCC49452), enteroaerogen (Enterococcus aerogenesATCC13048), vibrio cholerae (Vibrio cholera SJTU32001), Vibrio parahaemolyticus (Vibrioparahaemolyticus ATCC17802), Vibrio parahaemolyticus (Vibrio parahaemolyticusATCC33846), Vibrio vulnificus (Vibrio vulnficus ATCC27562), listeria monocytogenes (Listeria monocytogenes AB97021), listeria monocytogenes (Listeriamonocytogenes ATCC13313), sterilized water, M is DNA Marker.
Fig. 3 is the collection of illustrative plates result of PCR product after 1.5% agarose gel electrophoresis in implementation column 1.Swimming lane 1～44 is followed successively by: the rugged Crow promise of slope bacillus (ATCC29544), the rugged Crow promise of slope bacillus (BDCS001), the rugged Crow promise of slope bacillus (BDCS002), the rugged Crow promise of slope bacillus (BDCS003), the rugged Crow promise of slope bacillus (BDCS004), the rugged Crow promise of slope bacillus (BDCS005), the rugged Crow promise of slope bacillus (BDCS006), the rugged Crow promise of slope bacillus (BDCS007), the rugged Crow promise of slope bacillus (BDCS008), the rugged Crow promise of slope bacillus (BDCS009), the rugged Crow promise of slope bacillus (BDCS010), the rugged Crow promise of slope bacillus (BDCS011), the rugged Crow promise of slope bacillus (BDCS012), the rugged Crow promise of slope bacillus (BDCS013), the rugged Crow promise of slope bacillus (BDCS014), the rugged Crow promise of slope bacillus (BDCS015), the rugged Crow promise of slope bacillus (BDCS016), the rugged Crow promise of slope bacillus (BDCS017), the rugged Crow promise of slope bacillus (BDCS018), the rugged Crow promise of slope bacillus (BDCS019), the rugged Crow promise of slope bacillus (BDCS020), the rugged Crow promise of slope bacillus (BDCS021), the rugged Crow promise of slope bacillus (BDCS022), the rugged Crow promise of slope bacillus (BDCS023), the rugged Crow promise of slope bacillus (BDCS024), the rugged Crow promise of slope bacillus (BDCS025), the rugged Crow promise of slope bacillus (BDCS026), the rugged Crow promise of slope bacillus (BDCS027), the rugged Crow promise of slope bacillus (BDCS028), the rugged Crow promise of slope bacillus (BDCS029), the rugged Crow promise of slope bacillus (BDCS030), the rugged Crow promise of slope bacillus (BDCS031), the rugged Crow promise of slope bacillus (BDCS032), the rugged Crow promise of slope bacillus (BDCS033), the rugged Crow promise of slope bacillus (BDCS034), the rugged Crow promise of slope bacillus (BDCS035), the rugged Crow promise of slope bacillus (BDCS036), the rugged Crow promise of slope bacillus (BDCS037), the rugged Crow promise of slope bacillus (BDCS038), the rugged Crow promise of slope bacillus (BDCS039), the rugged Crow promise of slope bacillus (BDCS040), the rugged Crow promise of slope bacillus (BDCS041), the rugged Crow promise of slope bacillus (BDCS042), sterilized water, M is DNA Marker.
Fig. 4 is that in embodiment 1, PCR product is verified the experimental patterns result of primer sensitivity through 1.5% agarose gel electrophoresis.Swimming lane 1～10 is followed successively by: 711.0ng/PCR, 71.1ng/PCR, 7.11ng/PCR, 711pg/PCR, 71.1pg/PCR, 7.11pg/PCR, 711fg/PCR, 71.1fg/PCR, 7.11fg/PCR, ddH
2o, M is DNA Marker.
Fig. 5 is the collection of illustrative plates result that in embodiment 2, PCR product is tested through 1.5% agarose gel electrophoresis checking artificial contamination.
Fig. 6 is the experimental patterns result that in embodiment 3, PCR product detects through 1.5% agarose gel electrophoresis checking actual sample.Wherein, swimming lane 1～30 is followed successively by: sample 1～sample 30, swimming lane 31～32:ddH
2o, the rugged Crow promise of slope bacillus (ATCC29544), M is DNA Marker.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, all according to ordinary method and condition, or selects according to catalogue.
In following embodiment, the primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Marker used is purchased from TIANGEN Biotech (Beijing) Co., Ltd., and product type is Marker I (MD101).
The detection of the 1 pair of rugged Crow promise of slope bacillus strain of embodiment
(1) adopt primer pair of the present invention and detection method to carry out PCR detection to the rugged Crow promise of slope bacillus reference culture (Cronobacter sakazakii) ATCC29544.
The right sequence of the primer is as follows:
SEN2-L：5’-ACTGGCTTGGGGCTAATA-3’(SEQ ID NO:1)，
SEN2-R：5’-AGAGGCGGATAAATCTTGT-3’(SEQ ID NO:2)。
Adopt above-mentioned primer pair, the genomic dna of the rugged Crow promise of the slope of take bacillus reference culture ATCC29544 is template, carry out foundation and the optimization of PCR reaction system and response procedures, through single factor, multifactorial experiment and cross experiment, thereby set up reaction system and the reaction parameter that is applicable to detect the rugged Crow promise of slope bacillus strain.
Described PCR reaction system is: 1 * PCR reaction buffer, 10-15mmol/L Mg
2+, 0.2-0.3mmol/L dNTP, 0.1-0.3 μ M primer SEN2-L, 0.1-0.3 μ M primer SEN2-R, Taq enzyme 0.05-0.1U/ μ L, DNA profiling 10-100ng/ μ L.Described pcr amplification program is: 94-95 ℃ of denaturation 3-5min, start afterwards following circulation, and the program of each circulation is: 94-95 ℃ of sex change 30-40s, 60-62 ℃ of annealing 30-40s, 68-72 ℃ is extended 30-40s; 30-35 altogether of circulation; After loop ends, 68-72 ℃ is extended 7-10min, is cooled to 4-15 ℃, finishes.Result shows, can obtain the amplified production of single 498bp within the scope of described reaction system and response procedures.
Optimum PCR reaction system is: 1 * PCR reaction buffer, 12.5mmol/L Mg
2+, 0.25mmol/L dNTP, 0.2 μ M primer SEN2-L, 0.2 μ M primer SEN2-R, Taq enzyme 0.04U/ μ L, DNA profiling 40ng/ μ L.Optimum pcr amplification program is: 94 ℃ of denaturation 5min, start afterwards following circulation, and the program of each circulation is: 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 35 of circulations; After loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.Under optimum reaction system and response procedures, its output at the amplified production of about 498bp is the highest, electrophoretic band the most obviously, the most clear.
Therefore, it is as follows that the present invention has set up optimum PCR detection method: first add 16.1 μ L sterilized waters in reaction tubes, then add successively 10 * PCR reaction buffer, 2.5 μ L, the Mg of 25mmol/L
2+2.0 μ L, the dNTP1.0 μ L of 2.5mmol/L, 5 μ M primer 1.0 μ L, 2.5U/ μ L Taq enzyme 0.4 μ L, finally adds template solution 2 μ L, and the system cumulative volume of making is 25 μ L, and using sterilized water as the negative control of template as reaction.Then after reaction tubes is centrifugal, put into PCR reaction instrument, according to following PCR program, carry out: at 94 ℃ of denaturation 5min, then do 35 circulations, the program of each circulation comprises 94 ℃ of sex change 30s, 63 ℃ of annealing temperatures, annealing time is 30s, then at 72 ℃, extends 30s, after loop ends, at 72 ℃, extends 10min, finally be cooled to 12 ℃, finish all operations program.
(2) Evaluation on specificity test
Get the rugged Crow promise of slope bacillus (Cronobacter sakazakii) reference culture and 42 strain food separation strains, and it is (as shown in table 1 to belong in Crow promise Bacillaceae (Cronobacter spp.) other several kinds except Cronobacter sakazakii, bacterial strain shown in table is that those skilled in the art can obtain by disclosed channel), according to genomic dna template extraction method, extract respectively genomic dna.Leaching process is as follows: got bacterial strain (as shown in table 1) is seeded to respectively in the TSB liquid nutrient medium of 5mL, at 37 ℃, cultivates after 8h, get 1mL bacterium liquid, put into 1.5mL centrifuge tube; At the centrifugal 10min of 5,000r/min, abandon supernatant liquor afterwards.With aseptic double-distilled water Eddy diffusion thalline, then at the centrifugal 5min of 12,000r/min, collect thalline.After centrifuge washing, add the aseptic ultrapure water of 100 μ L, in boiling water bath, boil 10min, take out immediately, at-20 ℃, place 10min.37 ℃ thaw after, the centrifugal 5min of 12,000r/min, get supernatant liquor place-20 ℃ standby.In this section, described food separation strain is taken from Infant Formula Enterprises or whey powder, first through API20E biochemical reagents bar, identify and belong to Crow promise Bacillaceae, then after the online BLAST comparison of these isolated strains basis after 16S rRNA order-checking, draw and belong to the rugged Crow promise of slope bacillus (Cronobacter sakazakii).
The DNA solution of every strain bacterial strain (DNA concentration 50ng/ μ L) is all got 2 μ L and is added in PCR reaction system and carry out amplified reaction as PCR reaction template, and reaction adopts the reaction system of aforementioned optimum and optimum amplification program.Adopt agarose gel electrophoresis to detect amplified production, whether judgement there is single amplified band in 498bp position, the results are shown in Table 1, and electrophoresis result is shown in Fig. 1～3.
As can be known from Table 1, except the reference culture and food separation strain of the rugged Crow promise of slope bacillus strain, all the other bacterial strains all do not have specific amplification band.In table 1 ,-: PCR result is negative; +: PCR result is positive.In table 1, Crow promise Bacillaceae (Cronobacter spp) has been used 8 strain reference cultures, has represented the typical standard bacterial strain of all kinds in this genus.In genus, comprise 6 kinds (wherein Cronobactersakazakii is called new combination), be the rugged Crow promise of slope bacillus ATCC29544, the positive Crow promise bacillus of malonate DSM18702, Mu Tingsi Crow promise bacillus ATCC51329, Crow, Zurich promise bacillus DSM18703, Crow promise bacillus gene kind 1NCTC9529 and Crow, Dublin promise bacillus.Wherein Crow, Dublin promise bacillus comprises three subspecies, i.e. promise bacillus Dublin, Crow, Dublin subspecies DSM18705, Crow, Dublin promise bacillus Lausanne subspecies DSM18706 and Crow, Dublin promise bacillus milk powder subspecies DSM18707.The sibship of slope rugged Crow promise Bacillaceae and enterobacter is nearest, especially with enterobacter in enterobacter cloacae sibship nearest.12 strains of enterobacter reference culture have been used in this test altogether, 3 strains of Shigella reference culture, 2 strains of serratia reference culture, Cray uncle belongs to reference culture 2 strains, 2 strains of Staphylococcus reference culture, 3 strains of Rhodopseudomonas reference culture, 2 strains of listeria reference culture, 3 strains of Vibrio reference culture and isolated strains 1 strain.These bacterial strains that use are all food-borne pathogens, and most enterobacteriaceae that all belongs to, and they and the rugged Crow promise of slope bacillus strain have nearer sibship.If these bacterial strains increase less than special fragment after primer of the present invention is tested by PCR, other is difficult to amplification to object fragment (498bp) more with the rugged Crow promise of slope bacillus strain sibship bacterial strain far away so, therefore through the systems biology experimental verification of the nearer bacterial strain of these sibships, fully guaranteed the specificity of detection method of the present invention and primer.The above results fully proves the method for the present invention rugged Crow promise of the slope bacillus (Cronobactersakazakii) of only increasing, for belonging to any bacterial classification in Crow promise Bacillaceae and other sibling species, all, without any amplification, therefore there is very high specificity.
Table 1. Evaluation on specificity bacterial strain uses therefor and test-results
(3) sensitivity evaluation test
Extract the genomic total DNA of slope rugged Crow promise bacillus (Cronobacter sakazakii), after measured, the concentration of the total DNA solution of the rugged Crow promise of slope bacillus gene group is 28.20 μ g/mL, with sterilized water, make 10 times of gradient dilutions, dilute altogether 10 gradients, each concentration gradient is got respectively 5 μ L and is added PCR reaction system, adopt the reaction system of aforementioned optimum and optimum amplification program to carry out PCR reaction, detected through gel electrophoresis amplified production, in gel imaging instrument, observe gel electrophoresis result, as shown in Figure 4, in figure: swimming lane 1～9:DNA template concentrations is respectively 711.0ng/PCR, 71.1ng/PCR, 7.11ng/PCR, 711pg/PCR, 71.1pg/PCR, 7.11pg/PCR, 711fg/PCR, 71.1fg/PCR, 7.11fg/PCR, swimming lane 10:ddH
2o, M:DNA marker.As shown in Figure 4, at the 6th swimming lane, can see band clearly, institute's corresponding DNA concentration is 7.11pg/PCR, and can't see amplified band after the 7th swimming lane.Therefore, judge that PCR detection sensitivity, as 7.11pg/PCR, has higher sensitivity.
Embodiment 2
The detection of the rugged Crow promise of artificial contamination's slope bacillus reference culture ATCC29544
The preparation of artificial contamination's sample: after sterilizing, getting respectively extent of dilution is 10 by the skimming milk solution of 100mL10%
-7, 10
-8with 10
-9each 1mL of the rugged Crow promise of slope bacillus ATCC29544 pure culture bacterium liquid (440cfu, 44cfu and 4.4cfu) add above-mentioned sterilized skimming milk solution.Place on shaking table, at 37 ℃, under 180r/min condition, cultivate, every 2h sampling 1 time, cultivate altogether 10h.Boiling method (Chen, W., S.Yu, C.Zhang, J.Zhang, C.Shi, Y.Hu, B.Suo, H.Cao, and X.Shi, 2011, Development of a single base extension-tag microarray for the detection ofpathogenic Vibrio species in seafood.Applied microbiology and biotechnology89 (6): 1979-1990.) extract genomic dna, adopt the reaction system of aforementioned optimum and optimum amplification program to carry out PCR reaction.Result as shown in Figure 5, by Fig. 5, can obviously be found out, the skimming milk solution of the rugged Crow promise of artificial access 440cfu slope bacillus ATCC29544 pure culture liquid can detect this bacterium through increasing after bacterium is cultivated 4h, and the skimming milk solution that manually accesses 44cfu and the rugged Crow promise of 4.4cfu slope bacillus ATCC29544 pure culture liquid can detect this bacterium through increasing after bacterium is cultivated 6h.
Embodiment 3
30 parts of food samples are all purchased from (comprising milk powder and vegetables etc.) market of farm produce and the large supermarket in Shanghai.Add 50g sample to 450mL buffered peptone water (buffer peptone water, BPW, pH8.0) in enrichment medium, increasing bacterium cultivates, after 37 ℃ of increasing bacterium 18h, every duplicate samples is got 1mL and is put into 1.5mL centrifuge tube, and with boiling method (Chen, W., S.Yu, C.Zhang, J.Zhang, C.Shi, Y.Hu, B.Suo, H.Cao, and X.Shi, 2011, Development of a single base extension-tagmicroarray for the detection of pathogenic Vibrio species in seafood.Appliedmicrobiology and biotechnology89 (6): 1979-1990.) extract genomic dna, 12, after the centrifugal 5min of 000rpm/min, get 2.5 μ L supernatant liquors as pcr template, with sterilized water, make negative control, adopt the reaction system of aforementioned optimum and optimum amplification program to carry out PCR detection.Each experiment repeats 3 times.Simultaneously, sample increases after bacterium 18～24h, by National Standard Method GB4789.40-2010, carry out the rugged Crow promise of isolation identification slope bacillus, and according to online BLAST, compare after 16S rRNA order-checking, the sample result that belongs to the rugged Crow promise of slope bacillus (Cronobacter sakazakii) is contrasted with PCR method.As shown in Figure 6, the PCR detection method of setting up through the present invention detects, there are 7 duplicate samples object band (498bp) to be detected, and this 7 duplicate samples is also separated and identified the rugged Crow promise of slope bacillus strain through national standard method and order-checking, the method for this experiment foundation has extreme high reliability as can be seen here.
Claims (7)
1. a method for the rugged Crow promise of the detection slope bacillus (Cronobactersakazakii) of the diagnosis of non-disease or therapeutic purpose, is characterized in that, comprises the following steps:
(1) extract the genomic dna of testing sample;
(2) take the genomic dna of step (1) gained is template, and the primer pair with sequence as shown in SEQ ID NO.1 and SEQ ID NO.2 carries out PCR reaction; With
(3) existence of the single amplified production of 498bp in detecting step (2) gained reaction product.
2. the method for claim 1, is characterized in that, the reaction system of described PCR reaction comprises: 1 * PCR reaction buffer, 10-15mmol/L Mg
2+, 0.2-0.3mmol/L dNTP, the sequence of 0.1-0.3 μ mol/L is the primer as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, Taq enzyme 0.05-0.1U/ μ L, and DNA profiling 10-100ng/ μ L; The response procedures of described PCR reaction is: 1. 94～95 ℃, and 3～5min; 2. 94～95 ℃, 30～40s; 3. 60～62 ℃, 30～40s; 4. 68～72 ℃, 30～40s; Step is 2. to 4. totally 30～35 circulations; 5. 68～72 ℃, 7～10min; 6. 4～15 ℃ of preservations.
3. the method for claim 1, is characterized in that, the reaction system of described PCR reaction comprises: 1 * PCR reaction buffer, 12.5mmol/L Mg
2+, 0.25mmol/L dNTP, the sequence of 0.2 μ mol/L is the primer as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, Taq enzyme 0.04U/ μ L, and DNA profiling 40ng/ μ L; The response procedures of described PCR reaction is: 1. 94 ℃, and 5min; 2. 94 ℃, 30s; 3. 60 ℃, 30s; 4. 72 ℃, 30s; Step is 2. to 4. totally 35 circulations; 5. 72 ℃, 10min; 6. 12 ℃ of preservations.
4. detect a primer pair for slope rugged Crow promise bacillus (Cronobacter sakazakii), it is characterized in that, by sequence, the primer as shown in SEQ ID NO.1 and SEQ ID NO.2 forms for it.
5. detect a test kit for slope rugged Crow promise bacillus (Cronobacter sakazakii), it is characterized in that, it comprises primer pair as claimed in claim 4.
6. test kit as claimed in claim 5, is characterized in that, described test kit also comprises PCR damping fluid, Taq enzyme, dNTP solution and Mg
2+in one or more.
7. test kit as claimed in claim 6, is characterized in that, described test kit also comprises gene extraction agent and/or positive gene group DNA.
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| CN104862391A (en) * | 2015-05-07 | 2015-08-26 | 贝因美婴童食品股份有限公司 | Kit and application thereof as well as detection method |
| CN105950471A (en) * | 2016-05-31 | 2016-09-21 | 东北农业大学 | Method and application of rapidly acquiring Cronobacter on basis of immunomagnetic bead technology |
| CN110951898A (en) * | 2019-12-30 | 2020-04-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof |
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| CN103409535B (en) * | 2013-08-16 | 2015-09-30 | 中国检验检疫科学研究院 | Detect the primer pair of Cronobacter sakazakii, probe and method |
| CN104862393B (en) * | 2014-04-16 | 2017-08-25 | 南开大学 | A kind of nucleotides and its application to Cronobacter sakazakii O antigen-specifics |
| CN104593487B (en) * | 2014-12-15 | 2017-06-27 | 合肥工业大学 | It is a kind of to distinguish the PCR RFLP molecular typing methods that Cronobacter sakazakii belongs to not of the same race |
| CN104561295B (en) * | 2014-12-29 | 2017-11-24 | 南京农业大学 | A kind of purpose nucleotide sequence and detection kit and detection method for detecting the rugged Cronobacter sakazakii of slope |
| CN110760570B (en) * | 2015-09-02 | 2023-01-24 | 上海产业技术研究院 | Method, primer group and kit for rapid constant-temperature detection of cronobacter sakazakii |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978291A (en) * | 2012-12-25 | 2013-03-20 | 光明乳业股份有限公司 | Cronobacter sakazakii strain detecting method, kit and primer |
-
2013
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-
2014
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|---|---|---|---|---|
| CN102978291A (en) * | 2012-12-25 | 2013-03-20 | 光明乳业股份有限公司 | Cronobacter sakazakii strain detecting method, kit and primer |
Non-Patent Citations (1)
| Title |
|---|
| 周胜利 等: "基于rpoB 基因的阪崎克罗诺杆菌分子检测体系的建立及其应用", 《安全与环境学报》, vol. 12, no. 4, 31 August 2012 (2012-08-31), pages 158 - 161 * |
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| CN104862391A (en) * | 2015-05-07 | 2015-08-26 | 贝因美婴童食品股份有限公司 | Kit and application thereof as well as detection method |
| CN105950471A (en) * | 2016-05-31 | 2016-09-21 | 东北农业大学 | Method and application of rapidly acquiring Cronobacter on basis of immunomagnetic bead technology |
| CN105950471B (en) * | 2016-05-31 | 2020-07-14 | 东北农业大学 | Method for rapidly capturing Cronobacter on basis of immunomagnetic bead technology and application |
| CN110951898A (en) * | 2019-12-30 | 2020-04-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of 4 species in Cronobacter and rapid detection method thereof |
| CN110951898B (en) * | 2019-12-30 | 2022-05-20 | 广东省微生物研究所（广东省微生物分析检测中心） | New specific molecular target of 4 species in Cronobacter and rapid detection method thereof |
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