CN102978291A - Cronobacter sakazakii strain detecting method, kit and primer - Google Patents
Cronobacter sakazakii strain detecting method, kit and primer Download PDFInfo
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Abstract
The invention discloses a Cronobacter sakazakii strain detecting method and kit. The method comprises the following steps of: (1) extracting genome DNA (Deoxyribose Nucleic Acid) of a sample to be detected and carrying out PCR (Polymerase Chain Reaction) amplification by taking the genome DNA as a template and a Cronobacter sakazakii specific amplification primer pair as a primers, wherein one primer in the primer pair has the sequence shown in SEQ ID NO.1, and the other primer has the sequence shown in SEQ ID NO.2; and (2) detecting the existence of a single amplification product at a 438bp position. The method provided by the invention is short in Cronobacter sakazakii strain detecting time, capable of reducing the detecting cost and increasing the detecting efficiency, single in specificity, reliable in detection result and simple in result judgment. The invention provides a simple, rapid and sensitive Cronobacter sakazakii strain detecting method for the technical field of food safety detection, which has greater significance for food safety in China.
Description
Technical field
The present invention relates to the microorganism detection field, be specifically related to a kind of method and test kit and primer that detects Crow promise Bacillaceae bacterial strain.
Background technology
Crow promise Bacillaceae (Cronobacter spp) is the entozoic a kind of Grain-negative sporeless bacterium of humans and animals enteron aisle.As a kind of important food source property conditioned pathogen, Crow promise Bacillaceae bacterial strain can cause the infection that the baby is fatal, and lethality rate is up to 10%-80%.It can cause the serious clinical symptom of baby usually, for example brain purulence ulcer, meningitis, necrotizing enterocolitis and general septicemia.The report that is separated to the rugged Crow promise of slope bacillus strain in infant formula was once arranged.Crow promise Bacillaceae bacterial strain is the essential condition pathogenic bacterium by formula powder harm infantile health.There are the risk that has the infant formula of Crow promise Bacillaceae to infect Crow promise Bacillaceae bacterial strain by edible pollution in ewborn infant or premature infant.In the pollution section investigation, find that in the whole Infant Formula Enterprises technological process of production, the points of contamination that can detect Crow promise bacillus accounts for 31% of gross sample point.Therefore, be the basis of achieve effective control to Crow promise Bacillaceae bacterial strain Rapid identification with differentiating.This bacterium after being named and defined in 1980 first, again be divided into Crow promise bacillus in 2008 by a kind again and newly belonged to (Cronobacter spp.), the subordinate is divided into 5 novel species (wherein Cronobacter sakazakii is called new combination), 1 Crow promise bacillus gene kind (Genomospeciese) and 3 new subspecieses, although the classification of this bacterium and title become, but the Enterobacter sakazakii standard detecting method of program mode is still continued to use at present detection, identify with traditional biochemical reaction, need 18-24h, and can't divide into anyly, this obviously can not satisfy the demand that Crow promise Bacillaceae bacterial strain is detected.Aspect discriminating, although existing amplified fragment length polymorphism (Amplified fragment length polymorphismas, AFLP), the multiple reliable method such as the comparison of 16S rRNA complete sequence, DNA-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, lack at present high efficiency evaluation and discrimination method to Crow promise Bacillaceae bacterial strain.
Summary of the invention
The technical problem to be solved in the present invention is to overcome existing conventional art long defective detection time, and a kind of PCR detection method, nucleic acid and primer pair of Crow promise Bacillaceae bacterial strain is provided.Adopt detection method of the present invention to detect Crow promise Bacillaceae bacterial strain, detection time is short, and cost is low, more has practicality, and detected result is special, and the result judges simply.
Technical scheme of the present invention is as follows:
One of technical scheme of the present invention is: a kind of method that detects Crow promise Bacillaceae bacterial strain may further comprise the steps:
(1) extract the genomic dna of sample to be checked, take it as template, take Crow promise Bacillaceae specificity amplification primer to as primer, carry out pcr amplification, described primer centering, the sequence of a primer are shown in the SEQ ID NO.1, and the sequence of another primer is shown in the SEQ ID NO.2; With
(2) existence of the single amplified production in detection 438bp position.
According to the present invention, in the step (1), among the described PCR, what the reaction system of PCR was better is: 1 * PCR reaction buffer, 10-15mmol/L Mg
2+, 0.2-0.3mmol/L dNTP, 0.1-0.3 μ M primer 1,0.1-0.3 μ M primer 2, Taq enzyme 0.01-0.1U/ μ L, dna profiling 10-100ng/ μ L.Better is: 1 * PCR reaction buffer, 12.5mmol/L Mg
2+, 0.25mmol/L dNTP, 0.2 μ M primer 1(SEQ ID NO.1), 0.2 μ M primer 2 (SEQ ID NO.2), Taq enzyme 0.04U/ μ L, dna profiling 50ng/ μ L.
In the step (1), among the described PCR, what the amplification program of PCR was better is: 92-95 ℃ of denaturation 3-6min, begin afterwards following circulation, and the program of each circulation is: 92-95 ℃ of sex change 20-40s, 60-65 ℃ of annealing 20-40s, 70-74 ℃ is extended 20-40s; 30-40 altogether of circulation; After the loop ends, 70-74 ℃ is extended 8-12min, is cooled to 4-15 ℃, finishes.Better is: 94 ℃ of denaturation 5min, begin afterwards following circulation, and the program of each circulation is: 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 35 of circulations; After the loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
In the step (2), the determination methods of described detected result is as follows: in the result who detects, if there is the single amplified production of 438bp position, then illustrates and contain Crow promise bacillus Pseudomonas bacterial strain in the sample to be checked; If there is no the single amplified production of 438bp position does not then contain Crow promise Bacillaceae bacterial strain in the sample to be checked.
Detection method described in the step (2) can be the method for this area routine, and preferably the detected through gel electrophoresis method can be sepharose, also can be polyacrylamide gel.
Method of the present invention is particularly useful for detecting the detection of the Crow promise Bacillaceae bacterial strain in Crow promise the Bacillaceae bacterial strain, particularly milk powder in the food.
Two of technical scheme of the present invention is: a kind of test kit that detects Crow promise Bacillaceae bacterial strain, it comprises that a sequence is that the primer shown in the SEQ ID NO.1 and a sequence are the primer shown in the SEQ ID NO.2.Better, also comprise 10 * PCR damping fluid, Taq enzyme, dNTP solution and MgCl
2Described 10 * PCR damping fluid, Taq enzyme, dNTP solution and MgCl
2All as described in the routine of this area.
Three of technical scheme of the present invention is: a kind of primer of the Crow promise Bacillaceae bacterial strain that increases, its sequence are shown in SEQ ID NO.1 or shown in SEQ ID NO.2.
On the basis that meets this area general knowledge, above-mentioned each optimum condition, but arbitrary combination namely get the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available getting all.
Positive progressive effect of the present invention is: adopt detection method of the present invention to detect Crow promise Bacillaceae bacterial strain, detection time is short, has reduced testing cost; Improved detection efficiency; Detection method of the present invention has single specificity, and detected result is reliable, and the result judges simply.The present invention provides a kind of method of detection Crow promise Bacillus bacteria of Simple fast sensitivity for the food safety detection technical field, and the food safety of China is had larger meaning.
Description of drawings
Fig. 1 be among the embodiment 1 the PCR product through 1.5% agarose gel electrophoresis experimental result.Swimming lane 1～17 is followed successively by: the rugged Crow promise of slope bacillus ATCC29544, Mu Tingsi Crow promise bacillus ATCC51329, Salmonella typhimurium ATCC14023, Salmonella enteritidis ATCC13311, enterobacter cloacae ATCC13047, intestinal bacteria ATCC43889, intestinal bacteria ATCC25922, Proteus mirabilis ATCC12453, proteus vulgaris ATCC33420, citrobacter ATCC8090, klebsiella pneumoniae ATCC27336, klebsiella pneumoniae ATCC46114, Song Zhi Shi Shigellae CMCC51334, shigella dysenteriae CMCC51335, shigella flexneri ATCC51371, Pseudomonas aeruginosa CDCB32116,100bp DNA Marker.
Fig. 2 be in the implementation column 1 the PCR product through 1.5% agarose gel electrophoretogram.Swimming lane 1～17 is followed successively by: Bacillus cereus ATCC1220, smell Serratia ATCC33077, serratia marcescens ATCC14040, pseudomonas putida ATCC17485, Pseudomonas alcaligenes IQCC12604, streptococcus aureus ATCC29213, streptococcus aureus ATCC8095, faecium ATCC14025, enterococcus faecalis ATCC49452, enteroaerogen ATCC13048, vibrio cholerae SJTU32001, Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus ATCC33846, Vibrio vulnificus ATCC27562, listeria monocytogenes AB97021, listeria monocytogenes ATCC13313,100bp DNA Marker.
Fig. 3 be in the implementation column 1 the PCR product through 1.5% agarose gel electrophoretogram.Swimming lane 1～17 is followed successively by: the rugged Crow promise of slope bacillus ATCC29544, Mu Tingsi Crow promise bacillus ATCC51329, Crow promise bacillus gene kind NCTC9529, the positive Crow promise bacillus of malonate DSM18702, Crow, Zurich promise bacillus DSM18703, Crow, Dublin promise bacillus Dublin subspecies DSM18705, Crow, Dublin promise bacillus Lausanne subspecies DSM18706, Crow, Dublin promise bacillus milk powder subspecies DSM18707, 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 BDCS008,100bp DNA Marker.
That the PCR product is through 1.5% agarose gel electrophoretogram in Fig. 4 implementation column 1.Swimming lane 1～17 is followed successively by: 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 BDCS024,100bp DNA Marker.
Fig. 5 be in the implementation column 1 the PCR product through 1.5% agarose gel electrophoretogram.Swimming lane 1～8 is followed successively by: 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, ddH
2O, 100bp DNA Marker.
Fig. 6 is that the PCR product is verified primer sensitivity experiment collection of illustrative plates through 1.5% agarose gel electrophoresis in the implementation column 1.Swimming lane 1～10 is followed successively by: 100bp DNA Marker, and 141.0ng/PCR, 14.1ng/PCR, 1.41ng/PCR, 141pg/PCR, 14.1pg/PCR, 1.41pg/PCR, 141fg/PCR, 14.1fg/PCR,, ddH
2O.
Embodiment
Mode below by embodiment further specifies the present invention, but does not therefore limit the present invention among the described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example according to ordinary method and condition, or is selected according to catalogue.
Crow promise Bacillaceae bacterial strain PCR detection method
The primer (synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) of synthetic conserved sequence in can pcr amplification Crow promise Bacillaceae gyrB gene order, primer sequence is as follows:
SEN2-L：5’-ATGGATAAAGAGGGCTACAG-3’（SEQ?ID?NO:1），
SEN2-R：5’-CGCCTGATTCTTACGGTTAC-3’（SEQ?ID?NO:2）。
Adopt above-mentioned primer, take the genomic dna of Crow promise Bacillaceae reference culture as template, carry out foundation and the optimization of PCR reaction system and response procedures, through single factor, multifactorial experiment and cross experiment, find that following reaction system and response procedures can both obtain the amplified production about single 438bp.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 1,0.1-0.3 μ M primer 2, Taq enzyme 0.01-0.1U/ μ L, dna profiling 10-100ng/ μ L.Described pcr amplification program is: 92-95 ℃ of denaturation 3-6min, begin afterwards following circulation, and the program of each circulation is: 92-95 ℃ of sex change 20-40s, 60-65 ℃ of annealing 20-40s, 70-74 ℃ is extended 20-40s; 30-40 altogether of circulation; After the loop ends, 70-74 ℃ is extended 8-12min, is cooled to 4-15 ℃, finishes.And optimum reaction system and response procedures are as follows, and the output of the amplified production about its 438bp is the highest, and electrophoretic band is the most obvious clear.Optimum PCR reaction system is: 1 * PCR reaction buffer, 12.5mmol/L Mg
2+, 0.25mmol/L dNTP, 0.2 μ M primer, 1,0.2 μ M primer 2, Taq enzyme 0.04U/ μ L, dna profiling 40ng/ μ L.Optimum pcr amplification program is: 94 ℃ of denaturation 5min, begin afterwards following circulation, and the program of each circulation is: 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 35 of circulations; After the loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
Therefore, it is as follows that the present invention has set up optimum PCR detection method: add first 16.1 μ L sterilized waters in reaction tubes, add successively 10 * PCR reaction buffer, 2.5 μ L, the Mg of 25mmol/L again
2+2.0 μ L, the dNTP 1.0 μ L of 2.5mmol/L, 5 μ M primers, 1.0 μ L, 2.5U/ μ L Taq enzyme 0.4 μ L adds template solution 2 μ L at last, and take sterilized water as template as the reaction negative control.Then after reaction tubes is centrifugal, put into PCR reaction instrument, carry out according to following PCR program: 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 extends 30s at 72 ℃, extends 10min at 72 ℃ after the loop ends, be cooled at last 12 ℃, finish all operations program.
It is (as shown in table 1 to get Crow promise Bacillaceae reference culture 8 strains such as the positive Crow promise bacillus of slope rugged Crow promise bacillus and malonate and 30 strain isolated strains, bacterial strain shown in the 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: the 38 strain bacterial strains (as shown in table 1) such as the positive Crow promise bacillus of slope rugged Crow promise bacillus and malonate are seeded to respectively in the TSB liquid nutrient medium of 50mL, 37 ℃ increase bacterium 8h after, get 1mL bacterium liquid, put into the 1.5mL centrifuge tube; At the centrifugal 10min of 3,000r/min, get supernatant liquor afterwards, again at the centrifugal 5min of 12,000r/min, collect thalline.With aseptic double-distilled water Eddy diffusion thalline, add the aseptic ultrapure water of 100 μ L behind the centrifuge washing, in boiling water bath, boil 15min, take out immediately, place 30min at-20 ℃.37 ℃ thaw after, the centrifugal 5min of 12,000r/min, get supernatant liquor place-20 ℃ for subsequent use.
The dna solution of every strain bacterial strain (DNA concentration 50ng/ μ L) is all got 2 μ L and is added to as the PCR reaction template and carry out amplified reaction in the PCR reaction system.Adopt agarose gel electrophoresis to detect amplified production, judge whether there is single amplified band in the 438bp position, the results are shown in Table 1, electrophoresis result is seen Fig. 1-5.
As can be known from Table 1, except the reference culture and food separation strain of Crow promise Bacillaceae bacterial strain, all the other negative control bacterial strains all do not have the specific amplified band.In the table 1 ,-: PCR result is negative; +: PCR result is positive.Crow promise Bacillaceae (Cronobacter spp) has used 8 strain reference cultures in the table 1, has represented the typical standard bacterial strain of all kinds in this genus.Comprise 6 kinds (wherein Cronobacter sakazakii is called new combination) in the genus, be the rugged Crow promise of slope bacillus ATCC25944, 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 Crow promise Bacillaceae and enterobacter is nearest, especially with enterobacter in the 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, the 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.Employed these bacterial strains all are food-borne pathogens, and most enterobacteriaceae that all belongs to, and they and Crow promise Bacillaceae bacterial strain have nearer sibship.If these bacterial strains increase less than special fragment after primer of the present invention is by the PCR experiment, other is difficult to amplification to purpose fragment (438bp) more with Crow promise Bacillaceae bacterial strain sibship bacterial strain far away so, therefore through the checking of the nearer bacterial strain of these sibships, fully guaranteed the specificity of the primer of this test design.The above results sufficient proof method of the present invention any bacterial strain of Crow promise Bacillaceae that can increase, and other any bacterial strain of can not increasing.
Table 1. Evaluation on specificity bacterial strain uses therefor and test-results
After measured, the concentration of Crow promise Bacillaceae genome DNA solution is 28.20 μ g/mL, make 10 times of gradient dilutions with sterilized water, dilute altogether 10 gradients, each gradient is got respectively 5 μ L and is added the PCR reaction system, and the detected through gel electrophoresis amplified production is observed the gel electrophoresis result in the gel imaging instrument, as shown in Figure 6, among the figure: swimming lane 2～9:DNA template concentrations is respectively 141.0ng/PCR, 14.1ng/PCR, 1.41ng/PCR, 141pg/PCR, 14.1pg/PCR, 1.41pg/PCR, 141fg/PCR, 14.1fg/PCR; Swimming lane 10:ddH
2O; Swimming lane: 100bp molecular weight standard (DNA marker).As shown in Figure 6, can see clearly band at the 7th swimming lane, institute's corresponding DNA concentration is 1.41pg/PCR, and can't see amplified band after the eight lanes.Therefore, judge that the PCR detection sensitivity is 1.41pg/PCR, has higher sensitivity.
The detection of the rugged Crow promise of artificial contamination's slope bacillus reference culture ATCC25944
The preparation of artificial contamination's sample: the cow's milk of getting 10mL is with after the liquid TSB substratum of 89mL mixes, and adds the rugged Crow promise of the slope bacillus ATCC2594 pure culture bacterium liquid (7CFU/mL) of 1mL after the dilution, at 37 ℃, cultivates 12h under the 150r/min condition.Every 2h sampling 1 time, water-boiling method extracts genomic dna, carries out PCR by the condition after optimizing and detects.Utilize the PCR detection method of the Crow promise Bacillaceae bacterial strain of embodiment 1 foundation to carry out the PCR detection, can detect this bacterium behind the artificial contamination 8h.
Claims (9)
1. a method that detects Crow promise Bacillaceae bacterial strain is characterized in that, may further comprise the steps:
(1) extract the genomic dna of sample to be checked, take it as template, take Crow promise Bacillaceae specificity amplification primer to as primer, carry out pcr amplification, described primer centering, the sequence of a primer are shown in the SEQ ID NO.1, and the sequence of another primer is shown in the SEQ ID NO.2; With
(2) existence of the single amplified production in detection 438bp position.
2. the method for claim 1 is characterized in that, in the step (1), among the described PCR, the reaction system of PCR is: 1 * PCR reaction buffer, 10-15mmol/L Mg
2+, 0.2-0.3mmol/L dNTP, 0.1-0.3 μ M primer 1,0.1-0.3 μ M primer 2, Taq enzyme 0.01-0.1U/ μ L, dna profiling 10-100ng/ μ L.
3. method as claimed in claim 2 is characterized in that, in the step (1), among the described PCR, the reaction system of PCR is: 1 * PCR reaction buffer, 12.5mmol/L Mg
2+, 0.25mmol/L dNTP, 0.2 μ M primer, 1,0.2 μ M primer 2, Taq enzyme 0.04U/ μ L, dna profiling 40ng/ μ L.
4. the method for claim 1, it is characterized in that, in the step (1), among the described PCR, the amplification program of PCR is: 92-95 ℃ of denaturation 3-6min, begin afterwards following circulation, and the program of each circulation is: 92-95 ℃ of sex change 20-40s, 60-65 ℃ of annealing 20-40s, 70-74 ℃ is extended 20-40s; 30-40 altogether of circulation; After the loop ends, 70-74 ℃ is extended 8-12min, is cooled to 4-15 ℃, finishes.
5. method as claimed in claim 4 is characterized in that, in the step (1), among the described PCR, the amplification program of PCR is: 94 ℃ of denaturation 5min begin following circulation afterwards, the program of each circulation is: 94 ℃ of sex change 30s, and 63 ℃ of annealing 30s, 72 ℃ are extended 30s; Totally 35 of circulations; After the loop ends, 72 ℃ are extended 10min, are cooled to 12 ℃, finish.
6. the method for claim 1 is characterized in that, the detection method described in the step (2) is the agarose gel electrophoresis detection method.
7. a test kit that detects Crow promise Bacillaceae bacterial strain is characterized in that, it comprises that a sequence is that the primer shown in the SEQ ID NO.1 and a sequence are the primer shown in the SEQ ID NO.2.
8. test kit as claimed in claim 7 is characterized in that, described test kit also comprises 10 * PCR damping fluid, Taq enzyme, dNTP solution and MgCl
2
9. the primer of Crow promise Bacillaceae bacterial strain that increases is characterized in that, its sequence is shown in SEQ ID NO.1 or shown in SEQ ID NO.2.
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| CN103243171A (en) * | 2013-05-29 | 2013-08-14 | 光明乳业股份有限公司 | Method for detecting cronobacter sakazakii as well as kit and primer thereof |
| CN103409535A (en) * | 2013-08-16 | 2013-11-27 | 中国检验检疫科学研究院 | Primer pair, probe and method for detecting Cronobacter sakazakii |
| CN104561295A (en) * | 2014-12-29 | 2015-04-29 | 南京农业大学 | Target nucleotide sequence, detection kit and detection method for detecting cronobacter sakazakii |
| CN104593487A (en) * | 2014-12-15 | 2015-05-06 | 合肥工业大学 | PCR-RFLP molecular subtyping method for distinguishing different types of cronobacters |
| CN114182029A (en) * | 2021-11-30 | 2022-03-15 | 石家庄君乐宝乳业有限公司 | Primer combination and application thereof in detection of cronobacter sakazakii in dairy products |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243171A (en) * | 2013-05-29 | 2013-08-14 | 光明乳业股份有限公司 | Method for detecting cronobacter sakazakii as well as kit and primer thereof |
| CN103966353A (en) * | 2013-05-29 | 2014-08-06 | 光明乳业股份有限公司 | Detecting method for Cronobacter sakazakii as well as kit and primers used in detecting method |
| CN103409535A (en) * | 2013-08-16 | 2013-11-27 | 中国检验检疫科学研究院 | Primer pair, probe and method for detecting Cronobacter sakazakii |
| 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 |
| CN104593487A (en) * | 2014-12-15 | 2015-05-06 | 合肥工业大学 | PCR-RFLP molecular subtyping method for distinguishing different types of cronobacters |
| CN104561295A (en) * | 2014-12-29 | 2015-04-29 | 南京农业大学 | Target nucleotide sequence, detection kit and detection method for detecting cronobacter sakazakii |
| CN114182029A (en) * | 2021-11-30 | 2022-03-15 | 石家庄君乐宝乳业有限公司 | Primer combination and application thereof in detection of cronobacter sakazakii in dairy products |
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| CN102978291B (en) | 2014-12-24 |
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