CN109797232A - A kind of malonate Cronobacter sakazakii CRISPR classifying method - Google Patents
A kind of malonate Cronobacter sakazakii CRISPR classifying method Download PDFInfo
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Abstract
The invention discloses a kind of malonate Cronobacter sakazakii CRISPR classifying methods.This method carries out DNA sequencing by four kinds of CRISPR molecules to malonate Cronobacter sakazakii, and the intervening sequence in abstraction sequence combines the CRISPR type for determining malonate Cronobacter sakazakii according to the intervening sequence of several persons.The method of the present invention is simple, quickly, low cost, high resolution be in MLST classifying method, non-environmental-pollution, laboratory equipment is required low, and CRISPR molecule preserves all multi informations such as bacteriophage and the plasmid that history infects, can federated database realize the whole nation even global standards chemoattractant molecule trace to the source, be suitble to extend to the fields such as food, inspection and quarantine.
Description
Technical field
The invention belongs to molecular epidemiology fields, and in particular to a kind of parting side malonate Cronobacter sakazakii CRISPR
Method.
Background technique
The health of infant is always the emphasis of state and society concern, for the newborn of artificial feeding, milk powder and its
The safety of substitute food product is most important.Cronobacter sakazakii also known as Enterobacter sakazakii (Cronobacter (Enterobacter
Sakazakii it is)) a kind of strong pathogenic bacteria detected in milk powder, can lead to infant's meningitis, necrotizing enterocolitis
And bacteremia, the death rate are up to 40% to 80%, have caused the great attention in the whole world.It is now recognized that baby formula milk powder
Pollution Cronobacter sakazakii is the main channel of the infection of newborn bacterium, and international food and agricultural organization and the World Health Organization are Cronobacter bar
Pseudomonas is classified as the A class pathogenic bacteria in babies ' formula milk powder.However also occurs the infant infection Cronobacter bar of breast-feeding in the recent period
The Case Report of bacterium, even to this day, the host of the bacterium and propagation model are still unintelligible.Cronobacter sakazakii can also infect always simultaneously
Year people and the adult of hypoimmunity, symptom is relatively light, has Cronobacter sakazakii to cause the report of food poisoning in the recent period.Crow
Promise Bacillus includes 7 kinds, and malonic acid Cronobacter sakazakii (Cronobacter sakazakii) is that one of them important causes a disease
Kind.Prepared food as the result is shown is adjusted in the national food-borne pathogens pollution that we carry out early period, is sent out in the food such as vegetables and edible mushroom
The case where existing malonate Cronobacter sakazakii pollution, establish effective classifying method trace to the source for early molecule it is very necessary.
It is in recent years, with advances in technology, reproducible in addition to pulsed field gel electrophoresis (PFGE) technology high resolution,
It is known as except " goldstandard " of pathogenic microorganism molecule parting, other parting means based on electrophoresis have been lacked for disease
The molecule of opportunistic pathogen is traced to the source.The molecule that largely the parting means based on gene sequencing are applied to pathogen is traced to the source as widely used
Multisequencing classifying method (multi-locus sequence typing, MLST), core gene group multisequencing classifying method
(core genome multi-locus sequence typing, cgMLST), full-length genome multisequencing classifying method (whole
Genome multi-locus sequence typing, wgMLST) etc., the effect based on full-length genome parting be it is optimal,
But gene order-checking is at high cost, operator needs preferable bioinformatic analysis basis, and popularization and application have certain
Difficulty.
CRISPR-Cas system is that a kind of new prokaryotes defence alien gene group found in the recent period includes that bacteriophage enters
The effective immunologic mechanism invaded.CRISPR-Cas system is made of CRISPR molecule and cas gene.Due in CRISPR-Cas system
CRISPR molecule save bacterium by the Horizontal transfers form such as bacteriophage exogenous DNA molecule invade information, and
Retrospect bacterial genetic evolution laws there are other classifying methods can not look forward to by putting in order comprising temporal information for intervening sequence
And advantage, therefore have been used for the Genotyping of some pathogens and molecule is traced to the source in research, such as mycobacterium tuberculosis bacterium
The parting of strain, salmonella, yersinia pestis and campylobacter jejuni etc..
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of CRISPR classifying methods of malonate Cronobacter sakazakii.
The object of the present invention is to provide a kind of malonate Cronobacter sakazakiis of the diagnosing and treating purpose of non-disease
CRISPR classifying method, comprising the following steps:
A. the DNA of strain culture to be detected is extracted；
B. respectively using the DNA of step a as template, the PCR in the site CRISPR1, CRISPR2 and the site CRISPR3 is carried out
Amplification, wherein the amplimer in the site CRISPR1 is E-1F and E-1R, the amplimer in the site CRISPR2 be E-2F and E-2R,
The amplimer in the site CRISPR3 is E-3F and E-3R；PCR amplification is carried out to the site CRISPR3, if E-3F and E-3R are amplified
Band then plus expands the site CRISPR6, if not needing without band is amplified plus expanding the site CRISPR6, wherein the site CRISPR6
Amplimer be E-6F-mal and E-3R6R；
The primer E-1F, sequence as shown in SED ID NO.1,
The primer E-1R, sequence as shown in SED ID NO.2,
The primer E-2F, sequence as shown in SED ID NO.3,
The primer E-2R, sequence is as shown in SED ID NO.4；
The primer E-3F, sequence as shown in SED ID NO.5,
The primer E-3R, sequence is as shown in SED ID NO.6；
The primer E-6F-mal, sequence as shown in SED ID NO.7,
The primer E-3R6R, sequence is as shown in SED ID NO.8；
C. the pcr amplification product in the site CRISPR1, CRISPR2, CRISPR3 and CRISPR6 described in step b respectively
Carry out DNA sequencing；
D. the intervening sequence in CRISPR1, CRISPR2, CRISPR3 and CRISPR6 site sequence is extracted, according to interval sequence
The combination of column determines the CRISPR type of malonate Cronobacter sakazakii.
It is preferred that using the PCR amplification system of 50 μ L, wherein containingHS DNA Polymerase 0.5μ
The upstream primer of L, 5 × PrimeSTAR Buffer 10 μ L, dNTP 4 μ L, 10 μm of ol/L and downstream primer each 0.5 μ L, it is to be checked
Survey DNA template 1~2 μ L, polishing ddH of strain culture2O to 50 μ L；Amplification condition are as follows: 98 DEG C of initial denaturation 1min, then 98
DEG C 10s, 57~58 DEG C of 5s, 72 DEG C of 4min, totally 30 circulations；72 DEG C of extension 5min.
It is preferred that in the step b, if being detected after pcr amplification reaction using primer E-3F and E-3R without band, benefit
The site PCR amplification CRISPR3 is carried out with primer E-3F and E-3R6R.
It is preferred that the DNA sequencing of the step c, sequencing primer is the PCR amplification primer of step b.
Discovery targets the external sources such as bacteriophage and plasmid to the present inventor in malonate Cronobacter sakazakii CRISPR molecule
The intervening sequence of substance, illustrate in these sequences containing bacterial strain it is important infect information, can be applied to the bacterium infection genesis
Tracking and propagation model building, and can federated database realize the whole nation even global standards chemoattractant molecule trace to the source, can extend to
The fields such as food, inspection and quarantine.The method of the present invention is time saving, and economical, high resolution is easily operated, to laboratory equipment and software
It is required that it is low, and contain important time sequencing information, it traces to the source conducive to bacterial strain molecule.
It traces to the source for the pollution of malonate Cronobacter sakazakii in milk powder and the monitoring of the infant infection bacterium is that disease is pre-
One of the vital task of anti-control mechanism, but there are high-end parting equipment deficiency and technical staff's water for current disease control mechanism
Equal the problems such as irregular.Classifying method of the invention is since principle is simple, and operation is easy, low in cost, rapid reaction,
Efficiently, accurately, non-environmental-pollution, and it is not high to the equipment requirement in laboratory, therefore can widely be applied to food, examine
The fields such as quarantine, while the monitoring and early warning of disease can be carried out.
CRISPR classifying method of the invention has the advantage that
1. it is simple to operate, it is low for equipment requirements；
2. parting is at low cost, less than the one third of MLST parting cost；
3. having better resolution ratio compared with MLST classifying method；
4. since CRISPR intervening sequence has special time sequencing, so that the classifying method breaks up with bacterial genetic
And the advantage of the multiple information such as region, the molecular epidemiology for being particularly suited for illness outbreak or food safety affair are traced to the source.
Classifying method of the invention can be real in the laboratory and base's disease control unit for failing to be equipped with complex and expensive parting equipment
It applies, there is preferable popularization and application value.
Detailed description of the invention
Fig. 1 is the CRISPR parting schematic diagram of malonate Cronobacter sakazakii.
Fig. 2 is the PCR amplification electropherogram spectrum of malonate Cronobacter sakazakii CRISPR.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
The CRISPR classifying method of 1 64 plants of malonate Cronobacter sakazakiis of embodiment
(1) bacterial strain
This 64 plants of strain isolation using of experiment is completed from the milk powder in the multiple areas in China, edible mushroom and vegetables sample
Conventional biochemical identification and Molecular Detection, are determined as malonate Cronobacter sakazakii.
(2) DNA of bacteria extracts
Extract the gene of above-mentioned 64 plants of bacterial strains to be measured respectively using the bacterial genomes DNA extraction kit of Magen company
The DNA of group DNA, extraction are saved in -20 DEG C.
(3) primer synthesizes
The primer table of 1 CRISPR molecule of table amplification
Primer concentration used in PCR amplification is 10 μm of ol/L.
(4) PCR amplification
4.1 CRISPR1 expand PCR reaction system:
CRISPR1 expands PCR reaction condition are as follows: 98 DEG C of initial denaturation 1min, then 98 DEG C of 10s, 58 DEG C of 5s, 72 DEG C of 4min,
After totally 30 circulations, 72 DEG C of last extension 5min.
4.2 CRISPR2 expand PCR reaction system:
CRISPR2 expands PCR reaction condition are as follows: 98 DEG C of initial denaturation 1min, then 98 DEG C of 10s, 58 DEG C of 5s, 72 DEG C of 4min,
After totally 30 circulations, 72 DEG C of last extension 5min.
4.3 CRISPR3 expand PCR reaction system:
CRISPR3 expands PCR reaction condition are as follows: 98 DEG C of initial denaturation 1min, then 98 DEG C of 10s, 57 DEG C of 5s, 72 DEG C of 4min,
After totally 30 circulations, 72 DEG C of last extension 5min.
If 4.4 E-3F and E-3R amplify band, plus expand CRISPR6；If not needing amplification CRISPR6 without band,
Change upstream primer E-3F and E-3R6R amplification CRISPR3 into, system and condition are the same as 4.3.
CRISPR6 expands PCR reaction system:
CRISPR6 expands PCR reaction condition are as follows: 98 DEG C of initial denaturation 1min, then 98 DEG C of 10s, 57 DEG C of 5s, 72 DEG C of 4min,
After totally 30 circulations, 72 DEG C of last extension 5min.
After reaction, 5 μ L PCR reaction products are taken to carry out electrophoresis on 1% Ago-Gel, in gel imaging system
Upper observation result.Fig. 2 is the site the malonate Cronobacter sakazakii CRISPR electrophoretogram amplified, Marker DL5000, by
Fig. 2 is as it can be seen that the CRISPR site sequence in different strains is different in size.Genotyping result is different, and see Table 2 for details.
(5) pcr amplification product is sequenced
The sequencing of CRISPR molecule is carried out using the primer of amplification, sequencing task is completed by Hua Da gene.
(6) CRISPR parting
The CRISPR parting schematic diagram of malonate Cronobacter sakazakii is as shown in Figure 1.
The intervening sequence (spacer) in CRISPR site sequence is extracted using CRISPR finder, passes through repetitive sequence
(repeat) both forward and reverse directions for judging CRISPR molecule, the sequence rich in AT base is leader sequence, from most from the leader sequence
The spacer of distant place starts computation sequence number.It will be saved as after the spacer de-redundancy extracted in different CRISPR molecules respectively individually
Sequence library, each spacer assigns a serial number.The spacer of the CRISPR molecule of every plant of bacterium finds sequence library by blast
In correspondence serial number, fail the new spacer molecule for finding corresponding serial number, be then added to sequence library, assign new serial number.According to
The spacer permutation and combination of CRISPR molecule gives a particular number, wherein the CRISPR molecule lacked is denoted as number zero, most
The combination number for integrating CRISPR molecule afterwards determines the CRISPR type of malonate Cronobacter sakazakii.
The CRISPR genotyping result of 2 64 plants of malonate Cronobacter sakazakiis of table
The resolution ratio of the CRISPR parting of 2 malonate Cronobacter sakazakii of embodiment and MLST classifying method compared with
(1) MLST parting
Mainly having chosen seven house-keeping genes of malonate Cronobacter sakazakii carries out amplification sequencing, will be on gained sequence information
Reach special website (https://pubmlst.org/cronobacter/) obtain corresponding MLST type, specific experiment behaviour
The website MLST is seen as method.
The comparison of the resolution ratio of (2) two kinds of classifying methods
The MLST parting and CRISPR genotyping result of 3 malonate Cronobacter sakazakii of table compare
Resolution ratio evaluation Simpson diversity indices (D) quantization means, its calculation formula is: D=1- ∑ [nj (nj-
1)]/[N (N-1)], wherein nj indicates the bacterial strain quantity of jth banding pattern, and N indicates experimental strain sum.D value is bigger to illustrate resolution ratio
It is stronger.
The resolution ratio of two kinds of classifying methods is respectively as follows:
Multilocus sequence typing MLST:D=0.8953
CRISPR classifying method: D=0.9727
It can be seen that the high resolution of CRISPR classifying method is in MLST classifying method.
Simultaneously as shown in table 3,13 plants of malonate Cronobacter sakazakii MLST partings are ST7, and CRISPR parting can be by it
It is subdivided into CT12~CT15, seven kinds of types of CT17~CT19,11 plants of malonate Cronobacter sakazakii MLST partings are ST60,
CRISPR parting is further divided into tetra- kinds of types of CT23, CT24, CT25 and CT26, and 9 plants of malonate Cronobacter sakazakii MLST divide
Type is ST211, and CRISPR parting is further divided into tri- kinds of types of CT2, CT3 and CT4.Illustrate CRISPR classifying method compared with MLST
There is parting stronger molecule to trace to the source ability, and molecular epidemiology tracking when tracing to the source for illness outbreak or pollution has important
Meaning.
In conclusion CRISPR classifying method of the invention has the advantage that
1. it is simple to operate, it is low for equipment requirements；
2. average parting is at low cost；
3. having better resolution ratio compared with MLST classifying method；
4. since CRISPR intervening sequence has special time sequencing, so that the classifying method breaks up with bacterial genetic
And the advantage of the multiple information such as region, the molecular epidemiology for being particularly suited for illness outbreak or food safety affair are traced to the source.
Based on above-mentioned advantage, which can be in laboratory and the base's disease for failing to be equipped with complex and expensive parting equipment
It controls unit to implement, there is preferable popularization and application value.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Sequence table
<110>Guangdong Microbes Inst (microbiological analysis inspection center, Guangdong Province)
Huankai Microbes Tech Co., Ltd., Guangdong
<120>a kind of malonate Cronobacter sakazakii CRISPR classifying method
<160> 8
<170> SIPOSequenceListing 1.0
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<213>artificial sequence (Artificial Sequence)
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cctgacctgg taaacagagt agcg 24
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
cgatttccag acgtwcggcg ttaa 24
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
cagttragat ggtgtacycg cata 24
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aragggcagc cgrtctttaa caag 24
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gttgagctta aaccctcccc ttgc 24
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gtcagcggya ccttcagcag tt 22
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<400> 7
gctattagca cctgactgat gtacg 25
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<213>artificial sequence (Artificial Sequence)
<400> 8
caggcattcc ggtaatattc gctc 24
Claims (4)
1. a kind of malonate Cronobacter sakazakii CRISPR classifying method of the diagnosing and treating purpose of non-disease, feature exist
In, comprising the following steps:
A. the DNA of strain culture to be detected is extracted；
B. respectively using the DNA of step a as template, the PCR amplification in the site CRISPR1, CRISPR2 and the site CRISPR3 is carried out,
Wherein the amplimer in the site CRISPR1 is E-1F and E-1R, the amplimer in the site CRISPR2 be E-2F and E-2R,
The amplimer in the site CRISPR3 is E-3F and E-3R；PCR amplification is carried out to the site CRISPR3, if E-3F and E-3R are amplified
Band then plus expands CRISPR6, if not needing plus expanding the site CRISPR6, the wherein expansion in the site CRISPR6 without band is amplified
Increasing primer is E-6F-mal and E-3R6R；
The primer E-1F, sequence as shown in SED ID NO.1,
The primer E-1R, sequence as shown in SED ID NO.2,
The primer E-2F, sequence as shown in SED ID NO.3,
The primer E-2R, sequence is as shown in SED ID NO.4；
The primer E-3F, sequence as shown in SED ID NO.5,
The primer E-3R, sequence is as shown in SED ID NO.6；
The primer E-6F-mal, sequence as shown in SED ID NO.7,
The primer E-3R6R, sequence is as shown in SED ID NO.8；
C. the pcr amplification product in the site CRISPR1, CRISPR2, CRISPR3 and CRISPR6 described in step b carries out respectively
DNA sequencing；
D. the intervening sequence in CRISPR1, CRISPR2, CRISPR3 and CRISPR6 site sequence is extracted, according to intervening sequence
Combine the CRISPR type for determining malonate Cronobacter sakazakii.
2. the method according to claim 1, wherein the PCR amplification of the step b, amplification system are as follows: adopt
With the PCR amplification system of 50 μ L, wherein containingHS DNA Polymerase 0.5 μ L, 5 × PrimeSTAR
10 4 μ L of μ L, dNTP of Buffer, the upstream primer and each 0.5 μ L of downstream primer of 10 μm of ol/L, strain culture to be detected
DNA template 1~2 μ L, polishing ddH2O to 50 μ L；Amplification condition are as follows: 98 DEG C of initial denaturation 1min, then 98 DEG C of 10s, 57~58 DEG C
5s, 72 DEG C of 4min, totally 30 times circulation；72 DEG C of extension 5min.
3. the method according to claim 1, wherein in the step b, if utilizing primer E-3F and E-3R warp
Detection then carries out the site PCR amplification CRISPR3 using primer E-3F and E-3R6R without band after pcr amplification reaction.
4. the method according to claim 1, wherein the DNA sequencing of the step c, sequencing primer is step
The PCR amplification primer of rapid b.
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Cited By (2)
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| CN112795673A (en) * | 2021-02-09 | 2021-05-14 | 上海市质量监督检验技术研究院 | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof |
| 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 |
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| CN103981256A (en) * | 2014-04-15 | 2014-08-13 | 中国人民解放军疾病预防控制所 | Salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method |
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| CN103981256A (en) * | 2014-04-15 | 2014-08-13 | 中国人民解放军疾病预防控制所 | Salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method |
| CN105112519A (en) * | 2015-08-20 | 2015-12-02 | 郑州大学 | CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN112795673A (en) * | 2021-02-09 | 2021-05-14 | 上海市质量监督检验技术研究院 | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof |
| CN112795673B (en) * | 2021-02-09 | 2022-03-01 | 上海市质量监督检验技术研究院 | CRISPR (clustered regularly interspaced short palindromic repeats) detection method for Cronobacter in food and kit thereof |
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