CN109797232B - Clonobacterium malonate CRISPR typing method - Google Patents
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Abstract
The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) typing method of clonorbacterium malonate. According to the method, four CRISPR molecules of the Cronobacter malonate are subjected to DNA sequencing, spacer sequences in the sequences are extracted, and the CRISPR type of the Cronobacter malonate is determined according to the combination of the spacer sequences. The method is simple, rapid, low in cost, higher in resolution than an MLST typing method, free of environmental pollution and low in requirements for laboratory equipment, the CRISPR molecules store a plurality of information such as historically infected phages and plasmids, national and even global standardized molecule tracing can be realized by combining a database, and the method is suitable for being popularized to the fields of food, inspection and quarantine and the like.
Description
Technical Field
The invention belongs to the field of molecular epidemiology, and particularly relates to a CRISPR (clustered regularly interspaced short palindromic repeats) typing method for Clonobacterium malonate.
Background
The health of infants is always the key point of national and social concern, and the safety of milk powder and its substitute food is very important for artificially fed newborn infants. Cronobacter sakazakii (Cronobacter sakazakii) is a highly pathogenic bacterium detected in milk powder, which can cause infant meningitis, necrotizing enterocolitis and bacteremia, with mortality rate of 40% to 80%, and has attracted global high attention. Currently, Cronobacter contaminated by the infant formula is considered to be a main channel for newborn infants to infect the Cronobacter, and the International food and agriculture organization and the world health organization list the Cronobacter as the A-type pathogenic bacteria in the infant formula. However, recently, the case report of Cronobacter Crohn infection of breast-fed infants also appears, and the host and transmission model of the bacterium are not clear to date. Meanwhile, the Cronobacter sakazakii can infect the old and the immunocompromised adults, the symptoms are relatively mild, and the Cronobacter sakazakii has recently been reported to cause food poisoning. The genus Cronobacter contains 7 species, of which Cronobacter malonate (Cronobacter sakazakii) is an important pathogenic species. The national food-borne pathogenic bacteria pollution regulation result developed in the early stage shows that the conditions of malonates Cronobacter malonate pollution in foods such as cooked food, vegetables and edible fungi are found, and an effective typing method is very necessary for early molecular tracing.
In recent years, with the progress of technology, besides the high resolution and good repeatability of the Pulsed Field Gel Electrophoresis (PFGE) technology, which is known as the "gold standard" for pathogenic microorganism molecular typing, other electrophoretic-based typing methods have been used for the molecular tracing of pathogenic bacteria. A large number of typing methods based on gene sequencing are applied to molecular tracing of pathogenic bacteria, such as widely-applied multi-sequence typing Methods (MLST), core genome multi-sequence typing methods (cgMLST), genome-wide multi-sequence typing methods (whole genome multi-sequence typing, wmLgST) and the like, the effect based on genome typing is optimal, but the genome sequencing cost is high, operators need to have a better bioinformatics analysis basis, and the popularization and application are difficult to a certain extent.
The CRISPR-Cas system is a recently discovered new efficient immune mechanism for prokaryotes to defend foreign genomes, including phage invasion. The CRISPR-Cas system consists of CRISPR molecules and Cas genes. The CRISPR molecules in the CRISPR-Cas system store information of invasion of bacteria by exogenous DNA molecules in a gene level transfer form such as bacteriophage, and the arrangement sequence of the spacer sequences contains time information, so that the method has the advantages that other typing methods cannot achieve for tracing the genetic evolution law of the bacteria, and the method is used for genotyping and molecular tracing research of some pathogenic bacteria, such as the genotyping of Mycobacterium tuberculosis strains, salmonella, Yersinia pestis, Campylobacter jejuni and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a CRISPR typing method of Cronobacter malonate.
The invention aims to provide a malonates Cronobacter sakazakii CRISPR typing method for non-disease diagnosis and treatment, which comprises the following steps:
a. extracting DNA of a strain culture to be detected;
b. respectively taking the DNA in the step a as a template, and carrying out PCR amplification on CRISPR1, CRISPR2 locus and CRISPR3 locus, wherein amplification primers of CRISPR1 locus are E-1F and E-1R, and amplification primers of CRISPR2 locus are E-2F and E-2R, CRISPR3 locus are E-3F and E-3R; performing PCR amplification on the CRISPR3 locus, if a band is amplified by E-3F and E-3R, additionally amplifying the CRISPR6 locus, and if no band is amplified, not additionally amplifying the CRISPR6 locus, wherein amplification primers of the CRISPR6 locus are E-6F-mal and E-3R 6R;
the sequence of the primer E-1F is shown as SED ID NO.1,
the sequence of the primer E-1R is shown as SED ID NO.2,
the primer E-2F has the sequence shown in SED ID NO.3,
the sequence of the primer E-2R is shown as SED ID NO. 4;
the primer E-3F has the sequence shown in SED ID NO.5,
the sequence of the primer E-3R is shown as SED ID NO. 6;
the sequence of the primer E-6F-mal is shown as SED ID NO.7,
the primer E-3R6R has the sequence shown in SED ID NO. 8;
c. performing DNA sequencing on PCR amplification products of the CRISPR1, CRISPR2, CRISPR3 and CRISPR6 sites in the step b respectively;
d. and extracting the spacer sequences in the CRISPR1, CRISPR2, CRISPR3 and CRISPR6 site sequences, and determining the CRISPR type of the malonates Cronobacter malonate according to the combination of the spacer sequences.
Preferably, a 50. mu.L PCR amplification system is used, which contains
0.5 mu L of HS DNA Polymerase, 10 mu L of 5 × PrimeSTAR Buffer, 4 mu L of dNTP, 0.5 mu L of each of 10 mu mol/L upstream primer and 10 mu mol/L downstream primer, 1-2 mu L of DNA template of the culture of the strain to be detected, and the ddH is supplemented2O to 50 μ L; the amplification conditions were: pre-denaturation at 98 ℃ for 1min, then 10s at 98 ℃ and 57-58 DEG C5s, at 72 ℃ for 4min, for 30 cycles; extension at 72 ℃ for 5 min.
Preferably, in the step b, if no band is detected after PCR amplification reaction by using the primers E-3F and E-3R, PCR amplification is carried out on the CRISPR3 site by using the primers E-3F and E-3R 6R.
Preferably, the DNA sequencing primer of step c is the PCR amplification primer of step b.
The invention finds spacer sequences of exogenous substances such as targeted phage and plasmid in Cronobacter malonate CRISPR molecules, shows that the sequences contain important infection information of strains, can be applied to the construction of tracking and transmission models of infection sources of the strains, can be combined with a database to realize national and even global standardized molecule tracing, and can be popularized to the fields of food, inspection and quarantine and the like. The method is time-saving, economic, high in resolution, easy to operate, low in requirements on laboratory equipment and software, and favorable for tracing strain molecules, and contains important time sequence information.
Tracing the pollution source of the clonobacterium malonate in the milk powder and monitoring the infection of the clonobacterium malonate in infants are one of important tasks of a disease prevention and control mechanism, but the current disease control mechanism has the problems of insufficient high-end typing equipment, uneven levels of technicians and the like. The typing method of the invention has the advantages of simple principle, easy operation, low cost, quick reaction, high efficiency, accuracy, no environmental pollution and low requirement on equipment in a laboratory, so the typing method can be more widely applied to the fields of food, inspection and quarantine, and the like, and can monitor and early warn diseases.
The CRISPR typing method disclosed by the invention has the following advantages:
1. the operation is simple and convenient, and the requirement on equipment is low;
2. the typing cost is low and is less than one third of the MLST typing cost;
3. better resolution compared to MLST typing methods;
4. because the CRISPR spacer sequence has a special time sequence, the typing method has the advantages of multiple information such as bacterial genetic differentiation, regions and the like, and is more suitable for tracing the molecular epidemiology of disease outbreaks or food safety events.
The typing method can be implemented in laboratories and base-level disease control units which cannot be equipped with complex and expensive typing equipment, and has good popularization and application values.
Drawings
Fig. 1 is a schematic diagram of CRISPR typing of cronobacter malonate.
Fig. 2 is a PCR amplification electropherogram of cronobacter malonate CRISPR.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example CRISPR typing method of 164 strains of Cronobacter malonate
(1) Bacterial strains
The 64 strains used in the experiment are separated from milk powder, edible fungi and vegetable samples in a plurality of areas in China, the traditional biochemical identification and molecular detection are completed, and the strains are determined to be the malonates Cronobacter.
(2) Bacterial DNA extraction
The genomic DNA of the 64 strains to be tested was extracted using a bacterial genomic DNA extraction kit from magenta, and the extracted DNA was stored at-20 ℃.
(3) Primer synthesis
Table 1 primer tables for CRISPR molecular amplification
The concentration of primers used in PCR amplification was 10. mu. mol/L.
(4) PCR amplification
4.1 CRISPR1 amplification PCR reaction system:
the PCR reaction conditions of CRISPR1 amplification are as follows: pre-denaturation at 98 deg.C for 1min, then at 98 deg.C for 10s, at 58 deg.C for 5s, and at 72 deg.C for 4min, and after 30 cycles, final extension at 72 deg.C for 5 min.
4.2 CRISPR2 amplification PCR reaction system:
the PCR reaction conditions of CRISPR2 amplification are as follows: pre-denaturation at 98 deg.C for 1min, then at 98 deg.C for 10s, at 58 deg.C for 5s, and at 72 deg.C for 4min, and after 30 cycles, final extension at 72 deg.C for 5 min.
4.3 CRISPR3 amplification PCR reaction system:
the PCR reaction conditions of CRISPR3 amplification are as follows: pre-denaturation at 98 deg.C for 1min, then at 98 deg.C for 10s, at 57 deg.C for 5s, and at 72 deg.C for 4min, and after 30 cycles, final extension at 72 deg.C for 5 min.
4.4 if the E-3F and the E-3R amplify bands, the CRISPR6 is amplified; if no band exists, the CRISPR6 does not need to be amplified, and the upstream primers E-3F and E-3R6R are replaced to amplify CRISPR3, and the system and the conditions are the same as 4.3.
CRISPR6 amplification PCR reaction system:
the PCR reaction conditions of CRISPR6 amplification are as follows: pre-denaturation at 98 deg.C for 1min, then at 98 deg.C for 10s, at 57 deg.C for 5s, and at 72 deg.C for 4min, and after 30 cycles, final extension at 72 deg.C for 5 min.
After the reaction, 5. mu.L of the PCR reaction product was electrophoresed on 1% agarose gel and the result was observed on a gel imaging system. Fig. 2 is an electrophoresis diagram of CRISPR sites of the amplified malonates cronobacter, and Marker is DL5000, as can be seen from fig. 2, CRISPR site sequences in different strains are different in length. The results of typing were varied and are detailed in Table 2.
(5) Sequencing of PCR amplification products
And (3) carrying out CRISPR molecular sequencing by using the amplified primers, wherein the sequencing task is completed by the Huada gene.
(6) CRISPR typing
A CRISPR typing scheme of cronobacter malonate is shown in figure 1.
Extracting a spacer sequence (spacer) in a CRISPR locus sequence by using a CRISPR finder, judging the positive and negative directions of a CRISPR molecule through a repeat sequence (repeat), taking a sequence rich in AT base as a leader sequence, and calculating a sequence number from the spacer farthest away from the leader sequence. And (3) redundantly removing the spacers extracted from different CRISPR molecules and storing the spacers as independent sequence libraries, wherein each spacer is assigned with a serial number. And finding the corresponding serial number in the sequence library by the spacer of the CRISPR molecule of each strain through blast, and adding the spacer into the sequence library to assign a new serial number if a new spacer molecule with the corresponding serial number cannot be found. A specific number is given according to the spacer permutation combination of the CRISPR molecules, wherein the CRISPR molecules deleted are marked as number zero, and finally the CRISPR type of the clonobacterium malonate is determined by combining the combined numbers of the CRISPR molecules.
CRISPR typing results of Cronobacter malonate Table 264
Example 2 resolution comparison of CRISPR typing and MLST typing methods for Cronobacter malonate
(1) MLST typing
Seven housekeeping genes of the clonorbacterium malonate are mainly selected for amplification sequencing, and the obtained sequence information is uploaded to a special website (https://pubmlst.org/cronobacter/) The corresponding MLST type is obtained, and the specific experimental operation method is shown in an MLST website.
(2) Comparison of the resolution of two typing methods
TABLE 3 comparison of MLST and CRISPR typing results of Cronobacter malonate
The resolution evaluation is quantitatively expressed by a Simpson diversity index (D), and the calculation formula is as follows: d ═ 1- ∑ [ nj (nj-1) ]/[ N (N-1) ], where nj denotes the number of strains of the jth band type and N denotes the total number of experimental strains. A larger value of D indicates a stronger resolution.
The resolutions of the two typing methods are respectively:
multi-site sequence typing MLST: d-0.8953
CRISPR typing method: d-0.9727
It can be seen that the resolution of CRISPR typing methods is higher than MLST typing methods.
Meanwhile, as shown in table 3, 13 strains of the malonates cronobacter MLST are classified into ST7, CRISPR classification can be subdivided into seven types of CT 12-CT 15 and CT 17-CT 19, 11 strains of the malonates cronobacter MLST are classified into ST60, CRISPR classification is further classified into four types of CT23, CT24, CT25 and CT26, 9 strains of the malonates cronobacter MLST are classified into ST211, and CRISPR classification is further classified into three types of CT2, CT3 and CT 4. The CRISPR typing method has stronger molecular traceability than MLST typing, and has important significance for molecular epidemiological tracing during disease outbreak or pollution traceability.
In summary, the CRISPR typing method of the present invention has the following advantages:
1. the operation is simple and convenient, and the requirement on equipment is low;
2. the average parting cost is low;
3. better resolution compared to MLST typing methods;
4. because the CRISPR spacer sequence has a special time sequence, the typing method has the advantages of multiple information such as bacterial genetic differentiation, regions and the like, and is more suitable for tracing the molecular epidemiology of disease outbreaks or food safety events.
Based on the advantages, the typing method can be implemented in laboratories and basic level disease control units which cannot be equipped with complex and expensive typing equipment, and has good popularization and application values.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
GUANGDONG HUANKAI MICROBIAL SCI & TECH. Co.,Ltd.
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Claims (2)
1. A method for CRISPR typing of Clonobacterium malonate comprising the steps of:
a. extracting DNA of a strain culture to be detected;
b. respectively taking the DNA in the step a as a template, and carrying out PCR amplification on CRISPR1, CRISPR2 locus and CRISPR3 locus, wherein amplification primers of CRISPR1 locus are E-1F and E-1R, and amplification primers of CRISPR2 locus are E-2F and E-2R, CRISPR3 locus are E-3F and E-3R; performing PCR amplification on the CRISPR3 locus, if a band is amplified by E-3F and E-3R, additionally amplifying CRISPR6, and if no band is amplified, not additionally amplifying the CRISPR6 locus, wherein amplification primers of the CRISPR6 locus are E-6F-mal and E-3R 6R;
the sequence of the primer E-1F is shown as SED ID NO.1,
the sequence of the primer E-1R is shown as SED ID NO.2,
the primer E-2F has the sequence shown in SED ID NO.3,
the sequence of the primer E-2R is shown as SED ID NO. 4;
the primer E-3F has the sequence shown in SED ID NO.5,
the sequence of the primer E-3R is shown as SED ID NO. 6;
the sequence of the primer E-6F-mal is shown as SED ID NO.7,
the primer E-3R6R has the sequence shown in SED ID NO. 8;
c. performing DNA sequencing on PCR amplification products of the CRISPR1, CRISPR2, CRISPR3 and CRISPR6 sites in the step b respectively;
d. extracting spacer sequences in the CRISPR1, CRISPR2, CRISPR3 and CRISPR6 site sequences, and determining the CRISPR type of the malonates Cronobacter malonate according to the combination of the spacer sequences;
the PCR amplification of the step b has the following amplification system: using 50. mu.L of a PCR amplification system containing
HSDNA Polymerase 0.5 uL, 5 × PrimeSTAR Buffer 10 uL, dNTP 4 uL, upstream primer and downstream primer of 10 umol/L each 0.5 uL, DNA template of strain culture to be detected 1-2 uL, and ddH is supplemented2O to 50 μ L; the amplification conditions were: performing pre-denaturation at 98 ℃ for 1min, then performing pre-denaturation at 98 ℃ for 10s, at 57-58 ℃ for 5s, and at 72 ℃ for 4min for 30 cycles; extending for 5min at 72 ℃;
in the step b, if no band is detected after PCR amplification reaction by using the primers E-3F and E-3R, PCR amplification is carried out on the CRISPR3 locus by using the primers E-3F and E-3R 6R.
2. The method of claim 1, wherein the sequencing primer of the DNA sequencing of step c is the PCR amplification primer of step b.
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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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| 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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