CN104561295A - Target nucleotide sequence, detection kit and detection method for detecting cronobacter sakazakii - Google Patents
Target nucleotide sequence, detection kit and detection method for detecting cronobacter sakazakii Download PDFInfo
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Abstract
The invention discloses a target nucleotide sequence, a detection kit and a detection method for detecting cronobacter sakazakii. The target nucleotide sequence is as shown in the sequence SEQ ID NO.3. The kit comprises a PCR buffer, a MgCl2 solution, dNTP, a CS21-L primer, a CS21-R primer and TaqDNA polymerase. The detection method comprises the following steps: firstly, extracting genome DNA of a sample to be detected, secondly, by taking the extracted genome DNA as a template, performing PCR reaction on CS21 by using the CS21-L primer and the CS21-R primer, and finally, detecting whether a PCR product contains a single amplification product with the size of 152bp. The detection method has the advantages of short detection time, high anti-interference property, low cost, good specificity and high sensitivity, and has great significance on food security of China.
Description
Technical field
The present invention relates to food source pathogenic microorganism detection field, particularly relate to a kind of detect the rugged Cronobacter sakazakii of slope object nucleotide sequence and detection kit and detection method.
Background technology
The rugged Cronobacter sakazakii of slope (Cronobacter sakazakii) is a kind of food source property conditioned pathogen, is under the jurisdiction of Cronobacter sakazakii and belongs to (Cronobacter spp).This bacterium can cause the diseases such as meningitis, microbemia, necrotizing enterocolitis, and mortality ratio is up to 50%-80%, and survivor also will suffer the harm of neural system sequela all the life.The infant that its main harm crowd is hypoimmunity crowd, particularly premature infant, baby weight is on the low side.Thus the international food microbial standard council is decided to be the bacterium of " serious harm specific crowd, life-threatening or cause chronic substantive sequela ".The rugged Cronobacter sakazakii of slope is extensively present in occurring in nature, milk-product, fruit, vegetables, and this bacterium all once isolated by coarse cereals etc.In addition, the rugged Cronobacter sakazakii of slope to be also present in the various equipment of food factories and family on kitchen appliance, can cause the secondary pollution of food.The extensive existence of the rugged Cronobacter sakazakii of slope has seriously threatened the life and health of the mankind, and can accomplish to propagate for it effective control infected for the rapid detection of the rugged Cronobacter sakazakii of slope.
Cronobacter sakazakii is confirmed to be one for 2008 and newly belongs to, and ends and has 7 kinds till 2014: Cronobactersakazakii, Cronobacter dublinensis, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter universalis and Cronobacter condimentii.At present for detecting and identifying that the method for Cronobacter sakazakii is mainly based on physiological and biochemical test, but all there is the shortcomings such as sense cycle length, complex operation in these methods, is thus difficult to realize the quick diagnosis to contaminated samples.The detection method of existing PCR-based can confirm result in the short period, but these method majorities concentrate on detection Cronobacter sakazakii genus, can detect the then comparatively rare of the rugged Cronobacter sakazakii of slope.Therefore the current detection method for the rugged Cronobacter sakazakii of slope lacks and cannot satisfy the demands.
Summary of the invention
The object of the invention is to make up existing detection technique sense cycle long, complex operation, insufficient sensitivity etc. are not enough and provide a kind of detect the rugged Cronobacter sakazakii of slope object nucleotide sequence and detection kit and detection method.Described test kit is short for detection time, and immunity from interference is strong, has higher specificity and not by the impact of other kinds in belonging to.
Technical scheme
A kind of object nucleotide sequence detecting the rugged Cronobacter sakazakii of slope, this sequence comprises 152bp, and sequence is GGCAGCATGTCATTATCGGACGCCAGCGACACGCCGGTGTACGCGACACTGTCGAA GACTTTGGCGTCAGTATTTCCTTCGCCTATCATCAGCCGGCTTTTCAGCGTGCGAA TATCACGGGAGACGTGGCTGTAGACCGAATGCCACTGATG.
The rugged Cronobacter sakazakii detection method of a kind of slope, comprises the following steps:
(1) genomic dna of testing sample is extracted;
(2) genomic dna extracted with step (1) is for template, PCR reaction is carried out with CS21-L and CS21-R primer pair CS21, wherein CS21-L primer sequence is 5 '-GGCAGCATGTCATTATCGG-3 ', CS21-R primer sequence is 5 '-CATCAGTGGCATTCGGTCTA-3 ';
(3) detect in PCR primer whether there is the single amplified production that size is 152bp according to claim 1.
The rugged Cronobacter sakazakii detection method of described slope, in step (2), the condition of PCR reaction is 10 × PCR buffer, 10-15mM MgCl
2, 0.2-0.3mM dNTP, 10-100 μM primer CS21-L, 10-100 μM primer CS21-R, 1-10ng/ μ L genomic dna, 0.5-1U/ μ L Taq archaeal dna polymerase, all the other are with distilled water polishing; PCR response procedures is: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C-62 DEG C, 30s; 4. 72 DEG C, 30s-60s; 5. 72 DEG C, 7-10min; 6. 4 DEG C of preservations; Wherein 2. to 4. 30-35 circulation altogether.
The rugged Cronobacter sakazakii detection method of described slope, in step (2), the condition of PCR reaction is 10 × PCR buffer, 15mMMgCl
2, 0.2mM dNTP, 10 μMs of primer CS21-L, 10 μMs of primer CS21-R, 1ng/ μ L genomic dna, 1U/ μ L Taq archaeal dna polymerase, all the other are with distilled water polishing; PCR response procedures is: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C, 30s; 4. 72 DEG C, 30s; 5. 72 DEG C, 10min; 6. 4 DEG C of preservations; Wherein 2. to 4. totally 35 circulations.
The rugged Cronobacter sakazakii detection method of described slope, is detected PCR primer and is undertaken by agarose gel electrophoresis or polyacrylamide gel electrophoresis in step (3).
The rugged Cronobacter sakazakii detection kit of a kind of slope, comprising: PCR buffer, MgCl
2solution, dNTP, CS21-L primer: 5 '-GGCAGCATGTCATTATCGG-3 ', CS21-R primer: 5 '-CATCAGTGGCATTCGGTCTA-3 ' and Taq archaeal dna polymerase;
Further, the rugged Cronobacter sakazakii detection kit of described slope, comprising: 10 × PCR buffer, 10-15mM MgCl
2solution, 0.2-0.3mM dNTP, 10-100 μM of CS21-L primer: 5 '-GGCAGCATGTCATTATCGG-3 ', 10-100 μM of CS21-R primer: 5 '-CATCAGTGGCATTCGGTCTA-3 ' and 0.5-1U/ μ L Taq archaeal dna polymerase.
The rugged Cronobacter sakazakii detection kit of described slope, described test kit also comprises the DNA standard substance of the rugged Cronobacter sakazakii reference culture of slope containing object nucleotide sequence according to claim 1.
The decision principle of amplified production is in the present invention: after PCR reaction, pass through detected through gel electrophoresis, if the PCR reaction product of step (2) gained has corresponding product in 152bp position, then result of determination is positive, if the reaction product of gained does not have corresponding product at 152bp, then result is judged to be feminine gender.
The present invention is applicable to food samples, especially the detection of baby formula milk powder.
Beneficial effect
The rugged Cronobacter sakazakii detection kit of a kind of slope provided by the invention and detection method thereof, have fast, specificity is good, immunity from interference is strong, result judges simple advantage, simultaneously highly sensitive, empirical tests, genomic lowest detection lower limit is as 135fg μ L to utilize method provided by the invention to judge
-1, cell detection lower limit is 5.5 × 10
3cFU mL
-1.The present invention is for China's food safety tool important in inhibiting.
Accompanying drawing explanation
Fig. 1 is that in embodiment 1, primer pair CS21 belongs to interior six kinds for template with the rugged Cronobacter sakazakii of slope and carries out 2% agarose gel electrophoresis detected result of PCR reaction.In figure, M:DS
tM2000DNA marker; N: negative control (ddH
2o); Swimming lane 1 – 6 is followed successively by: the rugged Cronobacter sakazakii CICC21560 of slope, Mu Tingsi Cronobacter sakazakii CICC21563, malonate positive Cronobacter sakazakii DSM18702, Zurich Cronobacter sakazakii DSM18703, Dublin Cronobacter sakazakii DSM18705 and C.universalis NCTC9529.
Fig. 2 is the PCR primer 2% agarose gel electrophoresis result measuring genome detection sensitivity and cell detection sensitivity in embodiment 1.In figure, M:DS
tM2000DNA marker; N: negative control (ddH
2o); Figure A corresponds to genome detection sensitivity, and swimming lane 1-7 is corresponding different genes group DNA concentration respectively: 13.5ng μ L
-1, 1.35ng μ L
-1, 135pg μ L
-1, 13.5pg μ L
-1, 1.35pg μ L
-1, 135fg μ L
-1, 13.5fg μ L
-1; Figure B corresponds to the detection sensitivity of cell, and swimming lane 1-8 is corresponding cell concn 5.5 × 10 respectively
7cFU mL
-1, 5.5 × 10
6cFU mL
-1, 5.5 × 10
5cFU mL
-1, 5.5 × 10
4cFUmL
-1, 5.5 × 10
3cFU mL
-1, 5.5 × 10
2cFU mL
-1, 5.5 × 10
1cFU mL
-1, 5.5 × 10
0cFU mL
-1
Fig. 3 is the PCR primer 2% agarose gel electrophoresis detected result measuring immunity from interference in embodiment 1.In figure, M:DS
tM2000DNA marker; N: negative control (ddH
2o).In figure: the cell concn of the corresponding intestinal bacteria CMCC23657 of swimming lane 1-10 is respectively 10
8cFU mL
-1, 10
7cFU mL
-1, 10
6cFU mL
-1, 10
5cFU mL
-1, 10
4cFU mL
-1, 10
3cFU mL
-1, 10
2cFU mL
-1, 10
1cFU mL
-1, 10
0cFU mL
-1, 10
-1cFU mL
-1.
Fig. 4 is PCR primer 2% agarose gel electrophoresis detected result in embodiment 2.In figure, M:DS
tM2000DNA marker; The corresponding slope of figure A: swimming lane 1-7 rugged Cronobacter sakazakii initial inoculation amount is 5.5 × 10
1cFU mL
-1time sample time: 0h, 2h, 4h, 6h, 8h, 10h, 12h.Swimming lane 8-14 is 5.5 × 10 to corresponding slope rugged Cronobacter sakazakii initial inoculation amount
0cFU mL
-1time sample time: 0h, 2h, 4h, 6h, 8h, 10h, 12h.The corresponding slope of figure B: swimming lane 1-7 rugged Cronobacter sakazakii initial inoculation amount is 5.5 × 10
1cFU mL
-1time sample time: 0h, 2h, 4h, 6h, 8h, 10h, 12h.Swimming lane 8-14 is contrast, and its sample time is respectively: 0h, 2h, 4h, 6h, 8h, 10h, 12h.
Embodiment
Mode below by embodiment and accompanying drawing carrys out description and interpretation the present invention, but the present invention to have more than be limited in described scope of embodiments.In this example, all primers all pay Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's synthesis, the DNA standard molecular weight DS used
tM2000DNA marker purchases Dongsheng bio tech ltd, Guangzhou.
Embodiment 1: to the detection of slope rugged Cronobacter sakazakii reference culture CICC21560
(1) pair of primers CS21 of the present invention is adopted to carry out PCR detection to slope rugged Cronobacter sakazakii reference culture CICC21560.
The sequence of the primer to CS21 is as follows:
CS21-L：5’-GGCAGCATGTCATTATCGG-3’；(SEQ ID NO.1)
CS21-R：5’-CATCAGTGGCATTCGGTCTA-3’。(SEQ ID NO.2)
Corresponding pcr amplification product size is 152bp.
PCR reaction system used in the present invention is as follows:
Preferably reaction system is: 10 × PCR buffer, 10-15mM MgCl
2, 0.2-0.3mM dNTP, 10-100 μM primer CS21-L, 10-100 μM primer CS21-R, 1-10ng/ μ L genomic dna, 0.5-1U/ μ L Taq archaeal dna polymerase, all the other are with distilled water polishing.Preferably PCR reaction conditions is: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C-62 DEG C, 30s; 4. 72 DEG C, 30s; Step is 2. to 4. 30-35 circulation altogether; 5. 72 DEG C, 10min; 6. 4 DEG C of preservations.
Under preferably reaction conditions, all can obtain single, clearly 152bp size strip.
As follows by optimizing the better reaction conditions obtained: 10 × PCR buffer, 15mM MgCl
2, 0.2mM dNTP, 10 μMs of primer CS21-L, 10 μMs of primer CS21-R, 1ng/ μ L genomic dna 1 μ L, 1U/ μ L Taq archaeal dna polymerase, all the other are with distilled water polishing.Described PCR response procedures is: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C, 30s; 4. 72 DEG C, 30s; Step is 2. to 4. totally 30 circulations; 5. 72 DEG C, 10min; 6. 4 DEG C of preservations.
Under the better condition optimized, the 152bp pcr amplification product band obtained is the most clear, the brightest.
Therefore the PCR detection method set up of the present invention is as follows:
10 × PCR buffer 2.5 μ L is added successively, 15mM MgCl in PCR reaction tubes
22 μ L, 0.2mM dNTP 1 μ L, 10 μMs of primer CS21-L 1 μ L, 10 μMs of primer CS21-R 1 μ L, 1ng/ μ L genomic dna 1 μ L, 1U/ μ L Taq archaeal dna polymerase 1 μ L, all the other are with distilled water polishing, and cumulative volume is 25 μ L, and arranges positive control and negative control.After PCR reaction tubes being put into centrifuge mixing reaction system, put into PCR instrument, react according to following condition: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C, 30s; 4. 72 DEG C, 30s; Step is 2. to 4. totally 35 circulations; 5. 72 DEG C, 10min; 6. 4 DEG C of preservations.Pcr amplification product is detected by 2% agarose gel electrophoresis.Fig. 1 is the PCR detected result of CS21, and its stripe size is 152bp, high-visible.Therefore, the sequence fragment of this 152bp can as the object nucleotide sequence detecting the rugged Cronobacter sakazakii of slope, and this Target Nucleotide Sequence is GGCAGCATGTCATTATCGGACGCCAGCGACACGCCGGTGTACGCGACACTGTCGAA GACTTTGGCGTCAGTATTTCCTTCGCCTATCATCAGCCGGCTTTTCAGCGTGCGAA TATCACGGGAGACGTGGCTGTAGACCGAATGCCACTGATG.(SEQ ID NO.3)
(2) Evaluation on specificity test
By rugged for slope Cronobacter sakazakii reference culture CICC21560 and the strain of 6 strain slope rugged Cronobacter sakazakii food separation, and belong to other 5 kinds of Cronobacter sakazakii genus, be inoculated in LB substratum, in 37 DEG C, 180rpm incubated overnight; Get 1mL bacterium liquid in 1.5mL centrifuge tube, supernatant discarded after 12000rpm 2min; Extraction Methods of Genome conveniently extracts and purified genomic dna; Genomic dna be positioned over-20 DEG C for subsequent use.Six strain slopes rugged Cronobacter sakazakii food separation bacterial strain is distinguished to by Physiology and biochemistry qualification and is planted.Extracting genome DNA and the purifying of other bacteriums outside the Cronobacter sakazakii shown in table 1 belongs to are similar.Bacterial strain involved in the present invention all can be obtained by open approach.
Respectively get 1 μ L after the genomic dna of extraction being diluted to 10ng/ μ L and carry out PCR reaction as template.React complete after, whether 2% agarose gel electrophoresis detects pcr amplification product, have single bar to bring judged result to be negative or positive according at 152bp place.Detailed PCR the results are shown in Table 1.
Relate to 34 strain bacterial strains in table 1 altogether, Cronobacter sakazakii belongs to and amounts to 12 strains, wherein, reference culture 6 strain: the rugged Cronobacter sakazakii of slope, Dublin Cronobacter sakazakii, Mu Tingsi Cronobacter sakazakii, the positive Cronobacter sakazakii of malonate, Zurich Cronobacter sakazakii, C.universalis; The strain of slope rugged Cronobacter sakazakii strain isolated 6.The outer totally 22 strain bacterial strains of Ke Nuoluo Bacillaceae, wherein, intestinal bacteria 4 strain, salmonella 9 strain, micrococcus aureus 1 strain, Pseudomonas fluorescens 1 strain, Liszt belongs to 2 strains, Bacillus cereus 1 strain, subtilis 1 strain, enterobacter cloacae 1 strain, micrococcus luteus 1 strain, bacillus pumilus 1 strain.The PCR result that table 1 is tested for Evaluation on specificity, result shows that all the other PCR results are feminine gender except the rugged Cronobacter sakazakii reference culture of slope and food separation strain, and this shows that the detection method in the present invention has higher specificity.
Table 1: the bacterial strain that Evaluation on specificity uses and PCR result thereof
*:-expression PCR result is negative
+ represent that PCR result is positive
(3) sensitivity evaluation test
A, genome sensitivity determination
The rugged Cronobacter sakazakii CICC21560 reference culture of inoculation slope, in LB substratum, 37 DEG C, after 180rpm incubated overnight, extracts and purified genomic dna.The genomic dna extracted uses ultramicron nucleic acid determination instrument to measure its concentration for 13.5ng/ μ L.10 times of gradient dilution genomic dnas, dilute 7 gradients altogether.Get 1 μ L genomic dna from each dilution gradient and add the PCR reaction system that cumulative volume is 25 μ L, carry out PCR reaction.After PCR reaction terminates, 2% agarose gel electrophoresis detects pcr amplification product, and its result is as shown in Fig. 2-A.As can be seen from the figure, swimming lane 6 is observable lower limit, and the concentration of its correspondence is 135fg μ L
-1, therefore judge that genomic lowest detection lower limit is as 135fg μ L
-1, sensitivity is higher.
B, cell concn sensitivity determination
Inoculate slope rugged Cronobacter sakazakii reference culture CICC21560 in LB substratum, 37 DEG C, 10 times of gradient dilutions after 180rpm incubated overnight, and determine its cell concn by coating counting.From cell concn 10
7cFU mL
-1-10
0cFU mL
-1respectively get 10ml cell culture in 8 gradients, adopt water-boiling method to extract genome, its process is as follows: boiling water bath 10min is placed on-20 DEG C of 10min, centrifuging and taking supernatant, and supernatant is the crude extract of genomic dna.The PCR reaction system that the genomic dna crude extract getting each dilution gradient of 1 μ L adds 25 μ L carries out PCR reaction.After PCR reaction terminates, 2% agarose gel electrophoresis detects pcr amplification product, and its result as shown in fig. 2-b, can clearly be found out from figure, and swimming lane 5 is observable minimum lower limit, and corresponding cell concn is 5.5 × 10
3cFU mL
-1.Therefore, cell detection lower limit is 5.5 × 10
3cFU mL
-1, sensitivity is better.
(3) anti-interference test
Rugged for slope Cronobacter sakazakii standard C ICC21560 and intestinal bacteria reference culture CMCC23657 is seeded in LB substratum respectively, 37 DEG C, 180rpm incubated overnight.The method of 10 times of gradient dilutions and coating counting is used to calculate the cell concn of the rugged Cronobacter sakazakii CICC21560 of slope and intestinal bacteria CMCC23657.Get intestinal bacteria 10
8cFU mL
-1-10
-1cFUmL
-110 each 1ml of cell concn gradient and cell concn are 10
7cFU mL
-1slope rugged Crow bacillus 1mL mix respectively.Water-boiling method extracts the genomic dna of 10 compound samples, carries out PCR reaction using genomic dna as template.After PCR reaction terminates, 2% agarose gel electrophoresis detects pcr amplification product, its electrophoresis result as shown in Figure 3, as can be seen from the figure, along with the change of Bacillus coli cells concentration, PCR result all maintains consistence, and occurs without assorted band, and this illustrates that the detection method in the present invention has stronger immunity from interference.
Embodiment 2: the rugged Cronobacter sakazakii artificial contamination experiment of slope
Be seeded in LB substratum by rugged for slope Cronobacter sakazakii reference culture CICC21560,37 DEG C, 180rpm cultivates 12h.The cell concn of the rugged Cronobacter sakazakii of slope is calculated by gradient dilution and coating counting.Will wherein 10
1cFU mL
-1, 10
0cFUmL
-1with 10
-1cFU mL
-1add in the LB substratum of 89mL the 1mL cell culture of three cell concn gradients mixes (1ml physiological saline in contrast) with 10g baby formula milk powder after, 37 DEG C, 180rpm cultivates.From 0h to 12h, within every two hours, get a sample, preserve at being placed at-20 DEG C.Water-boiling method is used to extract genomic dna contained in sample.With carried genomic dna for template carries out PCR.2% agarose gel electrophoresis display (Fig. 4): be low to moderate 10 in inoculation
-1cFUmL
-1after the rugged Cronobacter sakazakii of slope, through the cultivation of 8h, can detect.
Embodiment 3: use the test kit based on primer pair CS21 to carry out actual sample detection
The Optimal system obtained according to optimizing reaction system in the present invention builds test kit, and carry out the detection of actual sample based on this, the PCR detection method exporting E. sakazakii in Milk Powder (Cronobacter sakazakii genus) method of inspection second section with SNT 1632.2-2013 contrasts.
Following reagent and enzyme is comprised: 10 × PCR buffer 1mL, 15mM MgCl in test kit constructed in the present invention
2800 μ L, 0.2mM dNTP 400 μ L, 10 μMs of primer CS21-L 400 μ L, 10 μMs of primer CS21-R 400 μ L, 1U/ μ L Taq archaeal dna polymerase 400 μ L.The theoretical access times of this test kit are 400 times.
The test kit operation instruction built in the present invention is as follows:
1. aseptically take sample 10g, add in 90mlLB substratum (other suitable enrichment medium also can), 37 DEG C, 180rpm cultivates 12h.
2. use water-boiling method or commercial reagent box to extract genomic dna.
3. carry out PCR according to following system (can in the light of actual conditions adjust):
4.2% agarose gel electrophoresis detects pcr amplification product, and in gel imaging system, observe whether there is the pcr amplification product that stripe size is 152bp, if exist, is the positive, does not exist, be then negative.
50 increment product all purchase the market of farm produce and the supermarket in Nanjing, carry out detecting and contrasting according to the operation instruction of test kit in the present invention and the PCR detection method of SNT1632.2-2013 outlet E. sakazakii in Milk Powder (Cronobacter sakazakii genus) method of inspection second section, final detected result is in table 2.
Table 2: in rower method and the present invention, test kit is to the detection of actual sample
*:-expression PCR result is negative
+ represent that PCR result is positive
As shown in table 2, rower SNT 1632.2-2013 method detects that 9 strain Cronobacter sakazakii belong to bacterial strain altogether, and test kit constructed in the present invention, detect the rugged Cronobacter sakazakii of 8 strain slope altogether.Both results are basically identical, and this illustrates that the test kit built in the present invention has higher reliability.The reason that two kinds of method detected results there are differences may be that rower method belongs to for detecting Cronobacter sakazakii, test kit of the present invention then for detecting the rugged Cronobacter sakazakii of slope, this also in realistic detection the rugged Cronobacter sakazakii of slope account for Cronobacter sakazakii belong to the overwhelming majority of strain isolated this be true.
Sequence table
<110> Agricultural University Of Nanjing
<120> mono-kind detects the object nucleotide sequence of the rugged Cronobacter sakazakii of slope and detection kit and detection method
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> CS21-L
<400> 1
ggcagcatgtcattatcgg 19
<210> 2
<211> 20
<212> DNA
<213> CS21-R
<400> 2
catcagtggcattcggtcta 20
<210> 3
<211> 152
<212> DNA
<213> aim sequence
<400> 3
ggcagcatgt cattatcgga cgccagcgac acgccggtgt acgcgacact gtcgaagact 60
ttggcgtcag tatttccttc gcctatcatc agccggcttt tcagcgtgcg aatatcacgg 120
gagacgtggctgtagaccgaatgccactgatg 152
Claims (8)
1. one kind is detected the object nucleotide sequence of the rugged Cronobacter sakazakii of slope, it is characterized in that, it is GGCAGCATGTCATTATCGGACGCCAGCGACACGCCGGTGTACGCGACACTGTCGAA GACTTTGGCGTCAGTATTTCCTTCGCCTATCATCAGCCGGCTTTTCAGCGTGCGAA TATCACGGGAGACGTGGCTGTAGACCGAATGCCACTGATG that this sequence comprises 152bp sequence.
2. the rugged Cronobacter sakazakii detection method of slope, is characterized in that, comprise the following steps:
(1) genomic dna of testing sample is extracted;
(2) genomic dna extracted with step (1) is for template, PCR reaction is carried out with CS21-L and CS21-R primer pair CS21, wherein CS21-L primer sequence is 5 '-GGCAGCATGTCATTATCGG-3 ', CS21-R primer sequence is 5 '-CATCAGTGGCATTCGGTCTA-3 ';
(3) detect in PCR primer whether there is the single amplified production that size is 152bp according to claim 1.
3. the rugged Cronobacter sakazakii detection method of slope according to claim 2, is characterized in that, in step (2), the condition of PCR reaction is 10 × PCR buffer, 10-15mM MgCl
2, 0.2-0.3mM dNTP, 10-100 μM primer CS21-L, 10-100 μM primer CS21-R, 1-10ng/ μ L genomic dna, 0.5-1 U/ μ L Taq archaeal dna polymerase, all the other are with distilled water polishing; PCR response procedures is: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C-62 DEG C, 30s; 4. 72 DEG C, 30s-60s; 5. 72 DEG C, 7-10min; 6. 4 DEG C of preservations; Wherein 2. to 4. 30-35 circulation altogether.
4. the rugged Cronobacter sakazakii detection method of slope according to claim 2, is characterized in that, in step (2), the condition of PCR reaction is 10 × PCR buffer, 15mM MgCl
2, 0.2mM dNTP, 10 μMs of primer CS21-L, 10 μMs of primer CS21-R, 1ng/ μ L genomic dna, 1 U/ μ L Taq DNA polymerase, all the other are with distilled water polishing; PCR response procedures is: 1. 94 DEG C, 5min; 2. 94 DEG C, 30s; 3. 60 DEG C, 30s; 4. 72 DEG C, 30s; 5. 72 DEG C, 10min; 6. 4 DEG C of preservations; Wherein 2. to 4. totally 35 circulations.
5. the rugged Cronobacter sakazakii detection method of slope according to claim 2, is characterized in that, detects PCR primer and undertaken by agarose gel electrophoresis or polyacrylamide gel electrophoresis in step (3).
6. the rugged Cronobacter sakazakii detection kit of slope, is characterized in that, comprising: PCR buffer, MgCl
2solution, dNTP, CS21-L primer: 5 '-GGCAGCATGTCATTATCGG-3 ', CS21-R primer: 5 '-CATCAGTGGCATTCGGTCTA-3 ' and Taq DNA polymerase.
7. the rugged Cronobacter sakazakii detection kit of slope according to claim 6, is characterized in that, comprising: 10 × PCR buffer, 10-15mM MgCl
2solution, 0.2-0.3mM dNTP, 10-100 μM of CS21-L primer: 5 '-GGCAGCATGTCATTATCGG-3 ', 10-100 μM of CS21-R primer: 5 '-CATCAGTGGCATTCGGTCTA-3 ' and 0.5-1 U/ μ L Taq DNA polymerase.
8. the rugged Cronobacter sakazakii detection kit of slope according to claim 6, is characterized in that, described test kit also comprises the DNA standard substance of the rugged Cronobacter sakazakii reference culture of slope containing object nucleotide sequence according to claim 1.
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| CN109593837A (en) * | 2017-09-29 | 2019-04-09 | 东北农业大学 | A method of the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope |
| 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 |
| CN112359122A (en) * | 2020-09-16 | 2021-02-12 | 山东省农业科学院农产品研究所 | Method for rapidly detecting cronobacter sakazakii in flammulina velutipes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109593837A (en) * | 2017-09-29 | 2019-04-09 | 东北农业大学 | A method of the nanogold colorimetric method based on magnetic capture quickly detects the rugged Cronobacter sakazakii of slope |
| 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 |
| CN112359122A (en) * | 2020-09-16 | 2021-02-12 | 山东省农业科学院农产品研究所 | Method for rapidly detecting cronobacter sakazakii in flammulina velutipes |
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