CN105950744A - Tracing method of cronobacter spp. in infant formula milk powder - Google Patents
Tracing method of cronobacter spp. in infant formula milk powder Download PDFInfo
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Abstract
The invention provides a tracing method of cronobacter spp. in infant formula milk powder. The method comprises the steps that by means of a multiple-locus sequence typing technology, molecular typing research is conducted on the cronobacter spp. of PIF raw materials, semi-finished products, finished products and processing environments thereof, population characteristics of the cronobacter spp. are revealed, a phylogenetic relationship of the cronobacter spp. is confirmed, phylogenetic analysis is conducted on the cronobacter spp. through bioinformatics, and therefore genetic characteristics of the cronobacter spp. in the above-mentioned environments are revealed systematically; on the basis of cronobacter spp. MLST typing, correlation analysis is conducted by taking ST of pathogenic bacteria and environmental sources as variables, key links which are most prone to pollute the cronobacter spp. in the PIF processing process and distribution rules are determined, and therefore the source of the cronobacter spp. in the infant formula milk powder is traced. The purpose of fundamentally monitoring the PIF production process and ensuring the PIF quality are achieved.
Description
Technical field
The invention belongs to field of food safety, review the pollution source of Cronobacter sakazakii in PIF and be specifically related to a kind of baby
The source tracing method of Cronobacter sakazakii in formula milk.
Background technology
Cronobacter sakazakii (Cronobacter spp.) be infant formula (powdered infant formula,
PIF) one of opportunistic pathogenic bacterium of most hazardness in, the neonate that body weight that baby is especially born in 28 days is lighter is taken in
After the PIF polluted by Cronobacter sakazakii, the pernicious diseases such as serious necrotizing enterocolitis, bacteremia and meningitis can be caused
Disease, mortality rate is up to 40%-80%.Cronobacter sakazakii is the kind that in enterobacteriaceae, environmental suitability is the strongest, thus while PIF
Processing environment (including the links such as supplementary material, pretreatment, spray drying, fluid bed and packaging) have been obtained for strict supervision
And control, but in presently commercially available PIF, the pollution rate of Cronobacter sakazakii is still up to 4.3%, it is seen that the pollution of these pathogenic bacterium is
Through having a strong impact on and constrain the sound development of China's PIF industry.
At present to the prevention and control of Cronobacter sakazakii in PIF mainly to the inspection of finished product with to raw materials for production, production environment
Supervision, the Inspection and Supervision method of this separation can not carry out prevention and control to Cronobacter sakazakii in PIF the most targetedly.
Summary of the invention
For the problems referred to above, the present invention provides the source tracing method of Cronobacter sakazakii in a kind of babies ' formula milk powder.
Cronobacter sakazakii is the genus that an emerging multiformity is abundant, at present at Cronobacter sakazakii Multilocus sequence typing number
According in storehouse (Multilocus Sequence Typing, MLST, http://www.pubmlst.org/cronobacter)
Comprising the Cronobacter sakazakii of 1090 strain separate sources, these bacterial strains are divided into 336 sequence type (sequence types, STs),
Which show the genetic diversity of its height.Meanwhile, the genetic diversity of Cronobacter sakazakii height adds it at poor environment
The probability of middle survival, more i.e. in population structure, dominant Cronobacter sakazakii more can adapt to bad environment, therefore
The genetic diversity of Cronobacter sakazakii in systematic research PIF and processing environment thereof, can review the Cronobacter bar in PIF finished product
Bacterium is originated, the more targeted prevention and control carrying out these pathogenic bacterium, thus formulates effective prevention and control measure.
The method utilizes Multilocus sequence typing technology, to being isolatable from the PIF supplementary material of same brand same batch, half one-tenth
The Cronobacter sakazakii of product, finished product and processing environment thereof carries out molecule parting research, on this basis, with ST and the ring of these pathogenic bacterium
Source, border carries out correlation analysis for variable, determines and the most easily pollutes the key link of Cronobacter sakazakii in the PIF course of processing and divide
Cloth rule, reaches to review the purpose of these pathogenic bacterium, thus realizes that Cronobacter sakazakii in PIF carries out from root specific aim and prevent
Control.
Step is:
1) sample will be gathered from PIF, PIF supplementary material of same brand same batch, semi-finished product, finished product and processing environment thereof
In the Cronobacter sakazakii that obtains, extract DNA, the 16SrRNA sequence of the DNA extracted expanded, to the 16SrRNA's obtained
PCR primer carries out gene sequencing, and the sequence obtained is compared in GENBANK data base, completes Molecular Identification；
2) specific amplification of house-keeping gene: the primer of synthesis house-keeping gene, carries out spy with Cronobacter sakazakii DNA for template
Specific amplification；
3) house-keeping gene order-checking: the PCR primer of Cronobacter sakazakii house-keeping gene is checked order, it is thus achieved that Cronobacter sakazakii is looked after the house base
The nucleotide sequence of cause；
4) determination of Cronobacter sakazakii MLST sequence type: log in Cronobacter sakazakii MLST data base, to every strain Cronobacter bar
The house-keeping gene allele of bacterium carries out sequence queries, it is thus achieved that the allele number of every strain bacterium, and determines according to allele number
The sequence type that Cronobacter sakazakii is final；
5) Source Tracing of Cronobacter sakazakii: utilize SPSS software, to be isolatable from the strain Cronobacter sakazakii of PIF processing environment
MLST sequence type and course of processing CCP be that variable carries out correlation analysis, obtain Cronobacter sakazakii MLST sequence type
And the dependency between CCP, thus review the pollution source of Cronobacter sakazakii, determine the pollution way of Cronobacter sakazakii
Footpath.
Concretely comprise the following steps:
1) sample will be gathered from PIF, PIF supplementary material of same brand same batch, semi-finished product, finished product and processing environment thereof
In the Cronobacter sakazakii that obtains, extract DNA, the 16SrRNA sequence choosing 27-F and the 1492-R primer DNA to extracting expands
Increasing, PCR amplification system is 10 × rTaq Buffer (containing 15mmol/L MgCl2) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, template
DNA2.0 μ L, 27-F1.0 μ L, 1492-R1.0 μ L, Taq archaeal dna polymerase 0.5 μ L, ddH2O36.5μL；Amplification condition is 95 DEG C
Denaturation 5min, 95 DEG C of degeneration 1min, 58 DEG C of annealing 30s, 72 DEG C extend 1min, circulate 30 times, and last 72 DEG C extend 5min；Profit
With agarose gel electrophoresis, amplified production is verified, and observation experiment result under UVP ultraviolet is phase system；Right
The PCR primer of 16SrRNA carries out gene sequencing, selects two-way survey to lead to, and the sequence obtained is carried out in GENBANK data base
Comparison, completes Molecular Identification；Wherein 27-F, 1492-R primer sequence is shown in SEQ ID NO:1, SEQ ID NO:2；
2) specific amplification of house-keeping gene: synthesis 7 to house-keeping gene atpD, fusA, glnS, gltB, gyrB, infB and
The primer of ppsA, primer sequence is shown in SEQ ID NO:3-SEQ ID NO:16, carries out specificity with Cronobacter sakazakii DNA for template
Amplification, the PCR amplification system of 7 pairs of primers is identical with condition, and PCR amplification system is that 10 × rTaq Buffer is (containing 15mmol/L
MgCl2) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, template DNA 5.0 μ L, forward primer 1.0 μ L, downstream primer 1.0 μ L, Taq DNA
Polymerase 1.0 μ L, ddH2O33.0μL；Amplification condition is 94 DEG C of denaturations 2min, 94 DEG C of degeneration 1min, 58 DEG C of annealing 1min, 72
DEG C extend 2min, circulate 30 times, last 72 DEG C extension 5min；Pcr amplification product is checked through 1% agarose gel electrophoresis, and
UVP ultraviolet gel is observed and recorded result under phase system；
3) house-keeping gene order-checking: the PCR primer sample presentation of house-keeping gene, purification, unidirectional survey are led to, obtained by Cronobacter sakazakii 7
Obtain the nucleotide sequence of 7 house-keeping genes of Cronobacter sakazakii；
4) determination of Cronobacter sakazakii MLST sequence type: log in Cronobacter sakazakii MLST data base, to every strain Cronobacter bar
7 allele of bacterium carry out sequence queries, it is thus achieved that the allele number of every strain bacterium, and determine gram according to 7 allele number
The sequence type that Luo Nuo bacillus is final；
5) Source Tracing of Cronobacter sakazakii: utilize SPSS software, to be isolatable from the Cronobacter sakazakii of PIF processing environment
MLST sequence type and course of processing CCP are that variable carries out correlation analysis, obtain Cronobacter sakazakii MLST sequence type with
Dependency between CCP, thus review the pollution source of Cronobacter sakazakii, determine the pollution channel of Cronobacter sakazakii.
Table 1 16S rRNA PCR amplification system and parameter
27 pairs of house-keeping gene amplification systems of table and amplification condition and parameter
Primer sequence in table 3 test
Accompanying drawing explanation
The PCR result of 7 house-keeping genes of Fig. 1；
The composition of Cronobacter sakazakii STs in Fig. 2 PIF and processing environment thereof；
7 house-keeping gene minimum likelihood phylogenetic trees of Fig. 3: Fig. 3 a:atpD gene minimum likelihood phylogenetic tree；Figure
3 b:fusA gene minimum likelihood phylogenetic trees；Fig. 3 c:glnS gene minimum likelihood phylogenetic tree；Fig. 3 d:gltB base
Because of minimum likelihood phylogenetic tree；Fig. 3 e:gyrB gene minimum likelihood phylogenetic tree；Fig. 3 f:infB gene minimum likelihood
Phylogenetic tree；Fig. 3 g:infB gene minimum likelihood phylogenetic tree；
Fig. 4 70 strain Cronobacter sakazakii maximum likelihood system grows tree；
Fig. 5 PIF processing environment and Cronobacter sakazakii sequence type correlation analysis.
Detailed description of the invention
Main agents:
Activation, purification and the qualification of embodiment 1 Cronobacter sakazakii
Strains tested is 80 strain Cronobacter sakazakii, and wherein 43 strains are isolatable from PIF, and 27 strains are isolatable from the processing environment of PIF, 4
Strain be Cronobacter sakazakii reference strain (C.sakazakii ATCC BAA-894, C.sakazakii ATCC 29004,
C.sakazakii ATCC 29544 and C.sakazakii ATCC 12868), 6 strains are Cronobacter sakazakii type strain
(C.malonaticus CDC105877T, C.dublinensis LMG23823T, C.turicensis LMG23827T,
C.universalis NCTC9529T, C.condimenti LMGT and C.muytjensii ATCC 51329T).In addition
E.aerogenes ATCC 13048, E.cloacae ATCC 35030, Escherichia CMCCB 44113 and
Escherichia O157 is as outer ginseng bacterial strain.Concrete condition is as shown in table 4, and above-mentioned strains tested is both from northeast agricultural
Key lab 503 of the dairy science Ministry of Education of university laboratory.
Table 4 strains tested and source thereof
Table 2-1 The strains and their sources
(1) activation of Cronobacter sakazakii
According to the proportional arrangement LB fluid medium of 25g (LB): 1000mL (distilled water), and high temperature at 121 DEG C is used to go out
Cool down after bacterium 30min, and be divided in sterile test tube stand-by according to the amount of 10mL.From cryogenic refrigerator, take out 80 strain glycerol preserve
Cronobacter sakazakii, first thaw in 4 DEG C of refrigerators, then return to room temperature in indoor.Inoculum concentration according to 1% by 100 μ L gram
Luo Nuo bacillus bacterium solution is inoculated in 10mLLB culture medium, cultivates 12h and reach the purpose of activation in 37 DEG C.
(2) purification of Cronobacter sakazakii
According to the proportional arrangement TSA culture medium of 40g (TSA): 1000mL (distilled water), use 121 DEG C, the condition of 30min
Lower high temperature sterilize, and in sterile petri dish, it is down flat plate, after cooling, Cronobacter sakazakii is carried out three rides.At 37 DEG C of incubators
Middle cultivation 24h, picking Cronobacter sakazakii list bacterium colony is also inoculated in LB fluid medium, cultivates 12h for 37 DEG C and obtains Cronobacter bar
The pure culture liquid of bacterium.
(3) extraction of Cronobacter sakazakii DNA
Draw Cronobacter sakazakii bacteria suspension 2mL after purification, use according to sky root bacterial genomes DNA extraction kit and say
The bright extraction that 80 strain Cronobacter sakazakii are carried out DNA, the DNA after extraction leave in-20 DEG C stand-by.
(4) Molecular Identification of Cronobacter sakazakii
According to the primer sequence in table 3, choose 27-F and the 1492-R primer 16SrRNA sequence to Cronobacter sakazakii DNA
Expanding, reaction system and amplification condition are shown in Table 5.Amplified production is verified by the agarose gel electrophoresis utilizing 1.0%,
And under UVP ultraviolet is phase system observation experiment result.Finally the PCR primer of Cronobacter sakazakii 16SrRNA is sent to Beijing promise
Match genome research centered finite company carries out gene sequencing, selects two-way survey to lead to, and by the sequence that obtains in GENBANK data
Comparing in storehouse, the Molecular Identification result completing Cronobacter sakazakii shows that 70 strain bacterial strains to be measured belong to Cronobacter sakazakii
Belonging to, similarity degree is all >=99%.
Table 5 16S rRNA PCR amplification system and parameter
The Multilocus sequence typing of embodiment 2 Cronobacter sakazakii
(1) 7 pair of house-keeping gene specific PCR amplification and gene sequencing
The specific amplification of (1) 7 pair of house-keeping gene
To the primer of house-keeping gene (atpD, fusA, glnS, gltB, gyrB, infB and ppsA), (gene order is shown in synthesis 7
Table 3), carry out specific amplification with Cronobacter sakazakii DNA for template, the PCR amplification system of 7 pairs of primers is identical with condition, such as table 6
Shown in.Pcr amplification product is checked through 1% agarose gel electrophoresis, and observed and recorded knot under UVP ultraviolet gel is phase system
Really, result is as shown in Figure 1.The purpose band of 7 house-keeping genes is clear, judges that its Product Sequence size is 7 according to pillar location
The purpose band of individual house-keeping gene.
67 pairs of house-keeping gene amplification systems of table and amplification condition and parameter
(2) gene sequencing of house-keeping gene
The PCR primer sample presentation of house-keeping gene, purification, unidirectional survey are led to by Cronobacter sakazakii 7, it is thus achieved that Cronobacter sakazakii 7
The nucleotide sequence of house-keeping gene.
(3) the MLST typing of Cronobacter sakazakii
(1) determination of Cronobacter sakazakii MLST sequence type
Log in Cronobacter sakazakii MLST data base (http://pubmlst.org/cronobacter/), select equipotential base
Because of sequence queries, input gene sequencing sequence, 7 allele of every strain Cronobacter sakazakii are carried out sequence queries, it is thus achieved that be every
The allele number of strain bacterium, clone's complexity (CC) and sequence type (ST), and sequence information is submitted in MLST data base
To every strain Cronobacter sakazakii identification code (ID) in Cronobacter sakazakii data base, result is as shown in table 7.
Table 7 70 strain Cronobacter sakazakii MLST information table
(1) allele
By data base's comparison, the allele number obtaining 7 house-keeping genes is as follows:
AtpD:atpD1, atpD3, atpD5, atpD10, atpD11, atpD16, atpD18, atpD19, atpD55,
atpD89；
FusA:fusA1, fusA7, fusA8, fusA11, fusA12, fusA13, fusA14, fusA15, fusA16,
fusA17、fusA37；
GlnS:glnS1, glnS3, glnS6, glnS7, glnS9, glnS10, glnS13, glnS16, glnS19,
glnS28、glnS37、glnS59、glnS107、glnS108；
GltB:gltB1, gltB3, gltB5, gltB8, gltB12, gltB15, gltB18, gltB19, gltB22,
gltB40、gltB41、gltB99、gltB109、gltB127、gltB128；
GyrB:gyrB1, gyrB5, gyrB8, gyrB9, gyrB10, gyrB11, gyrB15, gyrB16, gyrB18,
gyrB19、gyrB22、gyrB26、gyrB29、gyrB70、gyrB74、gyrB125；
InfB:infB1, infB3, infB5, infB14, infB15, infB17, infB20, infB24, infB35,
infB36、infB38、infB56、infB70；
PpsA:ppsA1, ppsA4, ppsA9, ppsA10, ppsA13, ppsA14, ppsA18, ppsA19, ppsA21,
ppsA23、ppsA26、ppsA32、ppsA80、ppsA102、ppsA160、ppsA161。
Wherein be found that 8 new allele, be respectively as follows: atpD89, glnS107, glnS108, gltB127,
GltB128, gyrB125, ppsA160 and ppsA161.
(2) determination of MLST sequence type
70 strain Cronobacter sakazakii have been divided into 19 sequence type, be respectively as follows: ST1, ST4, ST8, ST12, ST17, ST21,
ST22, ST31, ST40, ST50, ST64, ST83, ST201, ST258, ST259, ST268, ST260, ST269 and ST261, wherein
ST258, ST259, ST268, ST260, ST269 and ST261 are new sequence type, and ST201 and ST258 is positive gram of malonic acid
The sequence type of Luo Nuo bacillus.
(2) Phylogenetic Analysis of Cronobacter sakazakii based on MLST
7 house-keeping genes of splicing Cronobacter sakazakii, obtain the gene order of a length of 3036bp.Utilize Mega6.0 soft
70 strain Cronobacter sakazakii are carried out Phylogenetic Analysis by part, select maximum likelihood algorithm (Minimum likelihood
Algorithm), program repeats 1000 times, builds the phylogenetic tree of Cronobacter sakazakii, and selects C.sakazakii ATCC
BAA-894, C.sakazakii ATCC 29004, C.sakazakii ATCC 29544, C.sakazakii ATCC 12868,
C.malonaticus CDC105877T, C.dublinensis LMG23823T, C.turicensis LMG23827T,
C.universalis NCTC9529T, C.condimenti LMGTWith C.muytjensii ATCC 51329TAs reference and
Type strain.
The species characteristic of Cronobacter sakazakii in PIF and processing environment thereof is represented, as shown in Figure 2 by cake chart.
Slope rugged Cronobacter sakazakii 66 strain in 70 strain Cronobacter sakazakii, malonic acid positive Cronobacter sakazakii 4 strain, this explanation exists
In PIF and processing environment thereof, the rugged Cronobacter sakazakii of slope is the sociales that Cronobacter sakazakii belongs to.In terms of ST, the bacterial strain of ST4 is 18
Strain, accounts for the 27% of sum；The bacterial strain of ST1 is 14 strains, accounts for the 21% of sum；The bacterial strain of ST64 is 11 strains, accounts for the 16% of sum；It
Be afterwards ST12 (3 strains, 5%), ST21 (3 strains, 5%) and ST258 (3 strains, 5%), ST22 (2 strains, 3%), ST8 (2 strains, 3%),
ST261 (2 strains, 3%) and ST260 (2 strains, 3%)；Other 9 ST only have 1 strain Cronobacter sakazakii, be respectively as follows: ST17, ST31,
ST40, ST51, ST83, ST201, ST259, ST268 and ST260.The advantage of Cronobacter sakazakii in visible PIF and processing environment thereof
Sequence type is ST4, ST1 and ST64, and ST258 (3/4,75%) is malonic acid positive Cronobacter sakazakii ST that quantity is most in addition.
(4) Phylogenetic Analysis of Cronobacter sakazakii house-keeping gene
With E.aerogenes ATCC 13048, E.cloacae ATCC 35030, Escherichia CMCCB 44113
With Escherichia O157 as outer ginseng bacterial strain, utilize Mega6.0 software that different allele is carried out phylogeny and divide
Analysis, the maximum likelihood system obtaining 7 house-keeping genes grows tree, as shown in Figure 3.
Fig. 3 (a-g) has shown 7 house-keeping gene Phylogenetic Relationships clearly, and 7 house-keeping genes the most do not occur bright
Aobvious Horizontal Gene Transfer, these 7 house-keeping gene ratios of explanation are more conservative.The wherein phylogeny of atpD, fusA, gyrB and infB
Outer ginseng bacterial strain in tree is away from other sequences, and in glnS, E.cloacae ATCC 35030 and Cronobacter sakazakii have and close be
System growth relation, in gltB, E.cloacae ATCC 35030 and Cronobacter sakazakii have close Phylogenetic Relationships, in ppsA
E.aerogenes ATCC13048 and Cronobacter sakazakii have close Phylogenetic Relationships.
(5) Phylogenetic Analysis of Cronobacter sakazakii difference MLST sequence type
Minimum likelihood phylogenetic tree such as Fig. 4 institute of 70 strain Cronobacter sakazakii (there are 19 different MLST sequence type)
Show.C.sakazakii ATCC BAA-894, C.sakazakii ATCC 29004, C.sakazakii ATCC 29544 He
C.sakazakii ATCC 12868 is reference strain, C.malonaticus CDC105877T, C.dublinensis
LMG23823T, C.turicensis LMG23827T, C.universalis NCTC9529T, C.condimenti LMGTWith
C.muytjensii ATCC 51329TFor Cronobacter sakazakii type strain.
Fig. 4 shows Cronobacter sakazakii Phylogenetic Relationships clearly, and 7 kinds that Cronobacter sakazakii belongs to the most mutually maintain
A certain distance, and in these 7 kinds, the rugged Cronobacter sakazakii of slope and malonic acid positive Cronobacter sakazakii show closer to system
Relation.In slope rugged Cronobacter sakazakii kind, ST4 and ST258, ST64 and ST261 have closer to Phylogenetic Relationships；The third two
In the positive Cronobacter sakazakii kind of acid, the Phylogenetic Relationships of ST201 and ST7 (C.malonaticus CDC 105877) is nearer.And
According to the result of MLST data base's comparison, only differ a base between these close ST, owing to MLST typing can react whole
The Phylogenetic Relationships of individual genome, therefore ST4 and ST268, ST64 and ST261, the bacterial strain of ST201 and ST7 are likely to whole
The Phylogenetic Relationships of individual genome aspect is the most close.
(6) Source Tracing of Cronobacter sakazakii
Utilize SPSS software, to be isolatable from the MLST sequence type of 27 strain Cronobacter sakazakii of PIF processing environment and processed
Journey CCP (supplementary material, bar kill concentration after, be spray-dried after, fluid bed outlet, workshop finished product etc.) be that variable is carried out
Correlation analysis, obtains the dependency between Cronobacter sakazakii MLST sequence type and CCP, thus reviews Cronobacter bar
The pollution source of bacterium, determines the pollution channel of Cronobacter sakazakii, and result is as shown in Figure 5.
The Cronobacter sakazakii participating in analyzing amounts to 27 strains, is all isolatable from the production environment of certain PIF factory key production link
And in supplementary material and finished product, these detect that the CCP of Cronobacter sakazakii includes spray drying, fluid bed, fixing
Bed., fixed bed relevant to Cronobacter sakazakii ST17 and ST258 and Cronobacter sakazakii by correlation analysis discovery supplementary material
ST269 is relevant but the Cronobacter sakazakii of ST17, ST258 and ST269 type does not occur in the finished product of workshop, though therefore
On supplementary material and fixed bed, so it is isolated to Cronobacter sakazakii, but the two link is not the pollution way of Cronobacter sakazakii
Footpath；Meanwhile, ST1 is the most relevant to spray drying, fluid bed and workshop finished product, and ST64 is relevant to fluid bed and workshop finished product,
This explanation spray drying and fluid bed are the pollution channels of Cronobacter sakazakii；Additionally, ST4 and workshop finished product have dependency,
Although the Cronobacter sakazakii of ST4 type does not appears in processing link, but occurring in the finished product of workshop, this illustrates ST4
The Cronobacter sakazakii of type is the packaging link polluted product by PIF.
Claims (4)
1. the source tracing method of Cronobacter sakazakii in a babies ' formula milk powder, it is characterised in that: utilize Multilocus sequence typing skill
Art, carries out molecule parting research to the Cronobacter sakazakii of commercially available PIF, PIF raw material, semi-finished product, finished product and processing environment thereof, discloses
Its species characteristic, specifies the Phylogenetic Relationships of Cronobacter sakazakii, and by bioinformatics, Cronobacter sakazakii is carried out system
Developmental analysis, thus the inherited characteristic of Cronobacter sakazakii in the above-mentioned environment of announcement of system；At Cronobacter sakazakii MLST typing
On the basis of, carry out correlation analysis with ST and the environmental sources of these pathogenic bacterium for variable, determine in the PIF course of processing and the most easily pollute
The key link of Cronobacter sakazakii and the regularity of distribution, thus review the source of Cronobacter sakazakii in babies ' formula milk powder.
Method the most according to claim 1, it is characterised in that: step is:
(1) will gather in sample from PIF, PIF supplementary material of same brand same batch, semi-finished product, finished product and processing environment thereof
The Cronobacter sakazakii obtained, extracts DNA, expands, the 16SrRNA sequence of the DNA extracted to the 16SrRNA's obtained
PCR primer carries out gene sequencing, and the sequence obtained is compared in GENBANK data base, completes Molecular Identification；
(2) specific amplification of house-keeping gene: the primer of synthesis house-keeping gene, carries out special with Cronobacter sakazakii DNA for template
Property amplification；
(3) house-keeping gene order-checking: the PCR primer of Cronobacter sakazakii house-keeping gene is checked order, it is thus achieved that Cronobacter sakazakii house-keeping gene
Nucleotide sequence；
(4) determination of Cronobacter sakazakii MLST sequence type: log in Cronobacter sakazakii MLST data base, to every strain Cronobacter sakazakii
House-keeping gene allele carries out sequence queries, it is thus achieved that the allele number of every strain bacterium, and determines Crow according to allele number
The sequence type that promise bacillus is final；
(5) Source Tracing of Cronobacter sakazakii: utilize SPSS software, to be isolatable from the strain Cronobacter sakazakii of PIF processing environment
MLST sequence type and course of processing CCP are that variable carries out correlation analysis, obtain Cronobacter sakazakii MLST sequence type with
Dependency between CCP, thus review the pollution source of Cronobacter sakazakii, determine the pollution channel of Cronobacter sakazakii.
Method the most according to claim 1 and 2, it is characterised in that: concretely comprise the following steps:
1) obtain gathering in sample from PIF, PIF supplementary material of same brand same batch, semi-finished product, finished product and processing environment thereof
The Cronobacter sakazakii arrived, extracts DNA, and the 16SrRNA sequence choosing 27-F and the 1492-R primer DNA to extracting expands,
PCR amplification system is 10 × rTaq Buffer (containing 15mmol/L MgCl2) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, template
DNA2.0 μ L, 27-F1.0 μ L, 1492-R1.0 μ L, Taq archaeal dna polymerase 0.5 μ L, ddH2O36.5μL；Amplification condition is 95 DEG C
Denaturation 5min, 95 DEG C of degeneration 1min, 58 DEG C of annealing 30s, 72 DEG C extend 1min, circulate 30 times, and last 72 DEG C extend 5min；Profit
With agarose gel electrophoresis, amplified production is verified, and observation experiment result under UVP ultraviolet is phase system；Right
The PCR primer of 16SrRNA carries out gene sequencing, selects two-way survey to lead to, and the sequence obtained is carried out in GENBANK data base
Comparison, completes Molecular Identification；Wherein 27-F, 1492-R primer sequence is shown in SEQ ID NO:1, SEQ ID NO:2；
2) specific amplification of house-keeping gene: synthesis 7 is to house-keeping gene atpD, fusA, glnS, gltB, gyrB, infB and ppsA
Primer, primer sequence is shown in SEQ ID NO:3-SEQ ID NO:16, carries out specificity expansion with Cronobacter sakazakii DNA for template
Increasing, the PCR amplification system of 7 pairs of primers is identical with condition, and PCR amplification system is that 10 × rTaq Buffer is (containing 15mmol/L
MgCl2) 5.0 μ L, dNTP (2.5mM) 4.0 μ L, template DNA 5.0 μ L, forward primer 1.0 μ L, downstream primer 1.0 μ L, Taq DNA
Polymerase 1.0 μ L, ddH2O33.0μL；Amplification condition is 94 DEG C of denaturations 2min, 94 DEG C of degeneration 1min, 58 DEG C of annealing 1min, 72
DEG C extend 2min, circulate 30 times, last 72 DEG C extension 5min；Pcr amplification product is checked through 1% agarose gel electrophoresis, and
UVP ultraviolet gel is observed and recorded result under phase system；
3) house-keeping gene order-checking: the PCR primer sample presentation of house-keeping gene, purification, unidirectional survey are led to by Cronobacter sakazakii 7, it is thus achieved that gram
The nucleotide sequence of 7 house-keeping genes of Luo Nuo bacillus；
4) determination of Cronobacter sakazakii MLST sequence type: log in Cronobacter sakazakii MLST data base, 7 to every strain Cronobacter sakazakii
Individual allele carries out sequence queries, it is thus achieved that the allele number of every strain bacterium, and determines Cronobacter bar according to 7 allele number
The sequence type that bacterium is final；
5) Source Tracing of Cronobacter sakazakii: utilize SPSS software, to be isolatable from the MLST of the Cronobacter sakazakii of PIF processing environment
Sequence type and course of processing CCP are that variable carries out correlation analysis, obtain Cronobacter sakazakii MLST sequence type with crucial
Dependency between control point, thus review the pollution source of Cronobacter sakazakii, determine the pollution channel of Cronobacter sakazakii.
Method the most according to claim 3, it is characterised in that: described course of processing CCP is that supplementary material, bar kill
After concentration, be spray-dried after, fluid bed outlet, workshop finished product.
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| CN106480187A (en) * | 2016-10-13 | 2017-03-08 | 内蒙古农业大学 | Lactobacillus fermentum rapid tracing method, combined sequence used for method and construction method thereof |
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| CN113234839A (en) * | 2021-04-13 | 2021-08-10 | 天津科技大学 | Drying-resistant genotyping method for cronobacter sakazakii |
| CN113584195A (en) * | 2021-07-07 | 2021-11-02 | 东北农业大学 | Infant formula milk powder and method for tracing Cronobacter sakazakii in processing process of infant formula milk powder |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106480187A (en) * | 2016-10-13 | 2017-03-08 | 内蒙古农业大学 | Lactobacillus fermentum rapid tracing method, combined sequence used for method and construction method thereof |
| CN106480187B (en) * | 2016-10-13 | 2018-10-30 | 内蒙古农业大学 | Lactobacillus fermentum rapid tracing method, combined sequence used for method and construction method thereof |
| CN107245513A (en) * | 2017-04-27 | 2017-10-13 | 中国疾病预防控制中心传染病预防控制所 | For identifying that Cronobacter sakazakii belongs to primer pair group, probe, kit and the method for each " kind " |
| CN107245513B (en) * | 2017-04-27 | 2020-05-08 | 中国疾病预防控制中心传染病预防控制所 | Primer pair group, probe, kit and method for identifying various 'species' of Cronobacter |
| CN109295184A (en) * | 2018-08-28 | 2019-02-01 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of rugged Cronobacter sakazakii CRISPR classifying method of slope |
| CN109295184B (en) * | 2018-08-28 | 2021-06-15 | 广东省微生物研究所（广东省微生物分析检测中心） | Cronobacter sakazakii CRISPR typing method |
| WO2021115077A1 (en) * | 2019-12-09 | 2021-06-17 | 合肥工业大学 | Molecular typing method for distinguishing different strains of cronobacter by performing single restriction enzyme cutting on glua gene based on rsai |
| CN112094931A (en) * | 2020-09-24 | 2020-12-18 | 上海国际旅行卫生保健中心(上海海关口岸门诊部) | Primer group for amplifying MLST (MLST) typing and tracing housekeeping genes of Cronobacter sakazakii, second-generation sequencing and library building method and application |
| CN112094931B (en) * | 2020-09-24 | 2022-08-30 | 上海国际旅行卫生保健中心(上海海关口岸门诊部) | Primer group for amplifying MLST (MLST) typing and tracing housekeeping genes of Cronobacter sakazakii, second-generation sequencing and library building method and application |
| CN113234839A (en) * | 2021-04-13 | 2021-08-10 | 天津科技大学 | Drying-resistant genotyping method for cronobacter sakazakii |
| CN113584195A (en) * | 2021-07-07 | 2021-11-02 | 东北农业大学 | Infant formula milk powder and method for tracing Cronobacter sakazakii in processing process of infant formula milk powder |
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