CN110423833A - A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype - Google Patents
A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype Download PDFInfo
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Abstract
The invention discloses a kind of multiple PCR methods based on specific target identification Listeria monocytogenes serotype.This method comprises: Listeria monocytogenes bacterial strain to be measured is carried out Zengjing Granule, enrichment culture medium is obtained；The DNA for extracting enrichment culture medium, DNA extracting solution, PCR primer are uniformly mixed with PCR reactant, are carried out PCR reaction, are obtained PCR reaction product；By PCR reaction product electrophoresis, the serotype of detected bacterium is judged according to electrophoresis result.The present invention designs specific primer for specific target lmo2644, LMOf2365_0118, lmo1119, lmo0733 gene for the Listeria monocytogenes serotype newly excavated, and establishes the PCR identification method of Listeria monocytogenes serotype.These targets have the characteristics that accuracy height and specificity are good.This method has the characteristics that accuracy is high, specificity is good, identification speed is fast, low in cost and easy to operate.
Description
Technical field
The invention belongs to field of biotechnology, specifically relate to a kind of based on specific target identification Listeria monocytogenes serotype
Multiple PCR method.
Background technique
Listeria Monocytogenes (Listeria monocytogenes, LM) abbreviation Listeria monocytogenes are
A kind of food-borne pathogens of infecting both domestic animals and human.Listeria monocytogenes are widely present in nature, will lead in numerous food product
Pollution, and environmental pressure common in food processing process can be born.It is Listeria monocytogenes salt resistance alkali, cold-resistant, in 20% salt
It can still survive under concentration, it still can growth and breeding at 4 DEG C.Listeria monocytogenes are the unique human pathogens for causing listeriosis
Body, main infection the elderly, pregnant woman, children and immunologic hypofunction person.The clinical manifestation of listeriosis includes meningitis, brain
Scorching, miscarriage, heat generation gastroenteritis and septicemia etc., the death rate of listeriosis is up to 30%.Therefore, most countries are all right
Listeria monocytogenes in food carry out strict control, especially in ready-to-eat food.
It is reacted according to the serotype of thallus and flagellar antigen, Listeria monocytogenes can be divided into 13 serotypes, respectively
1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7 types.Wherein, this 4 kinds of blood of 1/2a, 1/2b, 1/2c, 4b
Clear type is main serotype, and 99% mankind's listeriosis is caused by the bacterial strain of these four serotypes.1/2a serotype is
Most commonly seen serotype in food, but the fulminant prevalence of listeriosis is mostly caused by bacterial strain as 4b serotype,
This illustrates that 4b serotype may have stronger virulence.Above 13 kinds of serotype can be further classified as 5 sero-groups
(serogroup), 1/2a and 3a type belongs to IIa groups, and 1/2b, 3b and 7 types belong to IIb groups, 1/2c and 3c type belongs to IIc groups, 4b,
4d and 4e type belongs to IVb groups, 4a and 4c type belongs to L groups.To the Serotype Identification of Listeria monocytogenes, in the prison of its epidemic strain
The aetology of survey, foodsafety assessment and epidemic outbreaks has important application value in tracing to the source.
Currently, being mainly that (inspection is examined in visible entry and exit to serotype method to the identification method of Listeria monocytogenes serotype
Epidemic disease professional standard SN-T 2521-2010), such method is needed using fimbrial antigen and somatic antigen, higher cost, and operation is multiple
It is miscellaneous, Yi Fasheng cross reaction, and qualification cycle is longer, completes primary identification needs 3 days or more time.
In existing patent, the PCR identification method of Listeria monocytogenes serotype, such as CN 107746890 A, CN
Target used in the patents such as 103602739 A is verified using Primer-BLAST, and discovery still lacks accuracy and special
Property all preferable Listeria monocytogenes serotype PCR identify primer；However, at present to the new PCR of Listeria monocytogenes serotype
Identify that the pertinent literature report of the excavation of target is less.To the excavation of its new PCR identification target and building for new PCR identification method
It is vertical, help to improve the accuracy and specificity of Listeria monocytogenes serotype PCR identification.
Summary of the invention
In order to overcome deficiencies of the prior art, the object of the present invention is to provide one kind based on specific target mirror
Determine the multiple PCR method of Listeria monocytogenes serotype.
The purpose of the present invention is realized at least through one of following technical solution.
A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype provided by the invention is
The PCR identification method for the Listeria monocytogenes that one species specificity is good, identification speed is fast, low in cost, easy to operate.
A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype provided by the invention, should
Sero-group IIa, IIc, L specificity target gene that PCR method uses is lmo2644, sero-group IVb, L specificity target gene
Specific target gene for LMOf2365_0118, sero-group IIc is lmo1119, Listeria monocytogenes specificity target gene
lmo0733。
The invention discloses the specific targets of one group of Listeria monocytogenes serotype and its corresponding Serotype Identification to draw
Object and identification method.The present invention for newly excavate Listeria monocytogenes serotype specific target lmo2644,
LMOf2365_0118, lmo1119, lmo0733 gene design specific primer, and establish the PCR of Listeria monocytogenes serotype
Identification method.The specificity target is excavated by comparing genomics and bioinformatics and is obtained, and it is accurate that this group of target has
The feature that degree is high and specificity is good.PCR Serotype Identification method of the invention has accuracy height, specific good, identification speed
Fastly, feature low in cost, easy to operate.
A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype provided by the invention, makes
Primer sequence are as follows:
Lmo2644-F:5 '-ACGGTATTACTTAGCGATGTGC-3 ',
Lmo2644-R:5 '-CAAACTCAAAATAAACGCTTCAA-3 ',
Its corresponding characteristic DNA segment is 669bp；
LMOf2365_0118-F:5 '-ACCGACCAATGTCTACACGC-3 ',
LMOf2365_0118-R:5 '-TTCACGCATTCTGGCATCT-3 ',
Its corresponding characteristic DNA segment is 889bp；
Lmo1119-F:5 '-GTGGTTCTGGTCTTGCCTTAG-3 ',
Lmo1119-R:5 '-GATTGAGAAAGAGGGTTGAAAA-3 ',
Its corresponding characteristic DNA segment is 243bp；
Lmo0733-F:5 '-AAAGCAATCAGAAAATCAAAAGG-3 ',
Lmo0733-R:5 '-GTACGATTTTATACGTTCTTCCGC-3 ',
Its corresponding characteristic DNA segment is 500bp.
A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype provided by the invention, packet
Include following steps:
(1) Zengjing Granule will be carried out in Listeria monocytogenes strain inoculated to be measured to culture medium (preferably TSB culture medium), obtain
To enrichment culture medium；
(2) genomic DNA for using RNA isolation kit extraction step (1) described enrichment culture medium, obtains DNA to be identified and mentions
Take liquid；
(3) by step (2) the DNA extracting solution to be identified, the PCR primer of the identification Listeria monocytogenes serotype
It is uniformly mixed with PCR reactant, obtains PCR reaction system, then carried out PCR reaction, obtain PCR reaction product；
(4) step (3) the PCR reaction product is subjected to agarose gel electrophoresis, electrophoresis result figure is obtained, if described
Occurs 669bp and 500bp band on electrophoresis result figure, then the serum of determination step (1) the Listeria monocytogenes bacterial strain to be measured
Group is IIa, and serotype is 1/2a or 3a；If only there is the band of 500bp, it is determined as sero-group IIb, i.e. serotype 1/
2b, 3b or 7；If only there is the band of 669bp, 243bp and 500bp, it is determined as sero-group IIc, i.e. serotype 1/2c or 3c；
If only there is the band of 889bp and 500bp, it is determined as sero-group IVb, i.e. serotype 4b, 4d or 4e；If only occur 669bp,
The band of 889bp and 500bp is then determined as sero-group L, i.e. serotype 4a or 4c；If without going out on the electrophoresis result figure
The now above band does not contain Listeria monocytogenes then in step (1) sample to be identified, be judged as Listeria monocytogenes yin
Property.
Further, step (1) described Zengjing Granule is the shaken cultivation in shaking table, and the temperature of the Zengjing Granule is 36
~38 DEG C, the time of Zengjing Granule is 8~12h, and the shaking speed of the Zengjing Granule is 210~230rpm.
Preferably, step (1) culture medium is TSB culture medium (i.e. pancreas junket soya peptone fluid nutrient medium).
Preferably, step (1) described Zengjing Granule is the shaken cultivation in shaking table, and the temperature of the Zengjing Granule is 37
DEG C, the time of Zengjing Granule is 10h, and the shaking speed of the Zengjing Granule is 220rpm.
Further, the DNA concentration in step (2) the DNA extracting solution to be identified is 10~500ng/ μ L.
Further, the PCR primer of step (3) the identification Listeria monocytogenes serotype includes PCR primer
Lmo2644, PCR primer LMOf2365_0118, PCR primer lmo0733 and PCR primer lmo1119.
Further, the PCR primer lmo2644 includes forward primer lmo2644-F and reverse primer lmo2644-R；
The forward primer lmo2644-F is as shown in SEQ ID NO:1, the reverse primer lmo2644-R such as SEQ ID
Shown in NO:2；
The PCR primer LMOf2365_0118 includes forward primer LMOf2365_0118-F and reverse primer
LMOf2365_0118-R；
The forward primer LMOf2365_0118-F is as shown in SEQ ID NO:3, the reverse primer LMOf2365_
0118-R is as shown in SEQ ID NO:4；
The PCR primer lmo1119 includes forward primer lmo1119-F and reverse primer lmo1119-R；
The forward primer lmo1119-F is as shown in SEQ ID NO:5, the reverse primer lmo1119-R such as SEQ ID
Shown in NO:6；
The PCR primer lmo0733 includes forward primer lmo0733-F and reverse primer lmo0733-R；
The forward primer lmo0733-F is as shown in SEQ ID NO:7, the reverse primer lmo0733-R such as SEQ ID
Shown in NO:8.
Further, step (3) the PCR reaction system includes based on volume parts:
5 parts of 10 × PCR reaction buffer；
1 part of dNTP solution；
1 part of PCR primer solution for identifying Listeria monocytogenes serotype；
1 part of DNA extracting solution to be identified；
1 part of Taq enzyme solution；
41 parts of water；
Wherein, the pH value of 10 × PCR reaction buffer is 8.2~8.4；The dNTP solution be dATP, dGTP,
The solution that tetra- kinds of deoxyribonucleoside triphosphate equal proportions of dTTP and dCTP are uniformly mixed with water, four in the dNTP solution
The concentration of kind deoxyribonucleoside triphosphate is 10~15mM；The PCR primer of the identification Listeria monocytogenes serotype is molten
Liquid is the solution identifying the PCR primer of Listeria monocytogenes serotype and being uniformly mixed with water, and the identification singly increases Liszt
The concentration of primer is each 8~12 μM in the PCR primer solution of bacterium serotype；The Taq enzyme solution is that Taq enzyme is uniformly mixed with water
Obtained solution, the concentration of the Taq enzyme solution are 2~3U/ μ L；The water is sterile and ultrapure water (DEPC without nucleotide
Water).
Preferably, the volume of step (3) described PCR system is 50 μ L.
Preferably, step (3) described PCR system (in terms of 50 μ L of volume) includes:
10 × PCR reaction buffer 5 μ L, dNTP 0.25mM, primer lmo2644-F, lmo2644-R, LMOf2365_
Each 0.2 μM of 0118-F, LMOf2365_0118-R, lmo1119-F, lmo1119-R, lmo0733-F, lmo0733-R, template DNA
1 μ L, Taq enzyme 2.5U the rest is DEPC water.
Further, the condition of step (3) the PCR reaction are as follows:
First in 95~98 DEG C of 2~4min of initial denaturation, it is then extended circulation, finally extends 4 under the conditions of 68~72 DEG C
~6min；
The Extended Cyclic are as follows: 14~16s is denaturalized under the conditions of 95~98 DEG C, anneal 14~16s under the conditions of 52~54 DEG C,
Extend 29~31s under the conditions of 68~72 DEG C, carries out 30~35 circulations.
Preferably, the condition of step (3) the PCR reaction are as follows:
First in 95 DEG C of initial denaturation 3min, it is then extended circulation, finally extends 5min under the conditions of 72 DEG C；
The Extended Cyclic are as follows: 15s is denaturalized under 95 DEG C of parts, anneal 15s under the conditions of 53 DEG C, extend 30s under the conditions of 72 DEG C,
Carry out 35 circulations.
Further, the mass percent concentration of step (4) described Ago-Gel is 1.5~3.0wt%.
Preferably, the agarose gel electrophoresis can take the PCR reaction product that volume is 5 μ L to carry out electrophoresis.
Compared with prior art, the invention has the advantages that and the utility model has the advantages that
(1) a kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype provided by the invention,
Its identification that Listeria monocytogenes serotype is carried out using PCR method, at low cost, the period is short, easy to operate, identification knot
Fruit is easy the characteristics of interpretation；
(2) a kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype provided by the invention,
Have the characteristics that accuracy height and high specificity, to the Serotype Identification of Listeria monocytogenes, especially in the prison of its epidemic strain
The aetology of survey, foodsafety assessment and epidemic outbreaks has important application value in tracing to the source.
Detailed description of the invention
Fig. 1 a is a kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype in embodiment 2
Evaluation on specificity part electrophoresis result figure；
Fig. 1 b is a kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype in embodiment 2
Evaluation on specificity another part electrophoresis result figure.
Specific embodiment
Specific implementation of the invention is described further below in conjunction with example, but implementation and protection of the invention is not limited to
This.If being that those skilled in the art can refer to prior art reality it is noted that there is the process of not special detailed description below
It is existing or understanding.Reagents or instruments used without specified manufacturer, being considered as can be by the commercially available conventional products being commercially available.
The PCR identification method that embodiment 1 establishes Listeria monocytogenes serotype is (described to be increased based on specific target identification list
The multiple PCR method of Listeria serotype)
It is compared by the whole genome sequence of the Listeria monocytogenes to different serotypes, screening obtains single increasing Li Si
Special bacterium sero-group IIa, IIc, L specificity target gene lmo2644, sero-group IVb, L specificity target gene LMOf2365_
The specific target gene lmo1119 of 0118, sero-group IIc, and to the full-length genome sequence of Listeria monocytogenes and other bacteriums
Column are compared, and screening obtains Listeria monocytogenes specificity target gene lmo0733.Use software Primer Premier 6
Design of primers is carried out, determines that primer sequence is as follows:
Lmo2644-F:5 '-ACGGTATTACTTAGCGATGTGC-3 ', as shown in SEQ ID NO:1；
Lmo2644-R:5 '-CAAACTCAAAATAAACGCTTCAA-3 ', as shown in SEQ ID NO:2；
LMOf2365_0118-F:5 '-ACCGACCAATGTCTACACGC-3 ', as shown in SEQ ID NO:3；
LMOf2365_0118-R:5 '-TTCACGCATTCTGGCATCT-3 ', as shown in SEQ ID NO:4；
Lmo1119-F:5 '-GTGGTTCTGGTCTTGCCTTAG-3 ', as shown in SEQ ID NO:5；
Lmo1119-R:5 '-GATTGAGAAAGAGGGTTGAAAA-3 ', as shown in SEQ ID NO:6；
Lmo0733-F:5 '-AAAGCAATCAGAAAATCAAAAGG-3 ', as shown in SEQ ID NO:7；
Lmo0733-R:5 '-GTACGATTTTATACGTTCTTCCGC-3 ', as shown in SEQ ID NO:8.
The primer of design is compared with the NT database of NCBI using online tool Primer-Blast, to verify this
The accuracy of invention identification primer.The 217 Listeria monocytogenes genomes of PCR identification primer of the invention in NT database
In, correctly identify the serotype that wherein 216 genomes correspond to bacterial strain.Using Chen Jianshun etc. (CN 103602739A), with
And the primer in the Lu Zhao invention of (CN 107746890A) such as newly carries out identical verifying, two inventions are distinguished can be correctly
207 correspond to the serotype of bacterial strain with 86 genomes in identification NR database, and as shown in table 1, table 1 is serotype of the invention
Identify the evaluation of the accuracy table of primer.Seen from table 1, Listeria monocytogenes Serotype Identification primer of the invention is compared to existing
Serotype Identification primer, have higher accuracy rate.
Table 1
Using above-described primer pair (PCR primer of the identification Listeria monocytogenes serotype), single increasing Lee is established
The PCR identification method of this special bacterium serotype.
The PCR reaction system total volume is 50 μ L, and the PCR reaction system includes 10 × PCR reaction buffer, 5 μ
L, 1 μ L of template DNA, the rest is DEPC water；Wherein contain dNTP0.25mM in PCR reaction system, primer lmo2644-F,
Lmo2644-R, LMOf2365_0118-F, LMOf2365_0118-R, lmo1119-F, lmo1119-R, lmo0733-F and
Each 0.2 μM of lmo0733-R, Taq enzyme 2.5U.
Affiliated PCR response procedures are as follows: 95 DEG C of initial denaturation 3min start amplification cycles, the program of each circulation later are as follows:
95 DEG C of denaturation 15s, 53 DEG C of annealing 15s, 72 DEG C of extension 30s carry out 35 circulations；Last 72 DEG C of extensions 5min.
The judgement of qualification result: take the PCR reaction product of 5 μ L in the Ago-Gel that mass percent concentration is 2wt%
Middle carry out electrophoresis, if only there is 669bp and 500bp band, the blood of determination step (1) the Listeria monocytogenes bacterial strain to be measured
Clear group is IIa, and serotype is 1/2a or 3a；If only there is the band of 500bp, it is determined as sero-group IIb, i.e. serotype 1/
2b, 3b or 7；If only there is the band of 669bp, 243bp and 500bp, it is determined as sero-group IIc, i.e. serotype 1/2c or 3c；
If only there is the band of 889bp and 500bp, it is determined as sero-group IVb, i.e. serotype 4b, 4d or 4e；If only occur 669bp,
The band of 889bp and 500bp is then determined as sero-group L, i.e. serotype 4a or 4c；If being determined as non-single increasing without the above band
Listeria.
A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype of the invention of embodiment 2
Evaluation on specificity
10 plants of single increasing Lee have been used altogether to the Evaluation on specificity of Listeria monocytogenes serotype PCR identification method of the invention
This special bacterium, the respectively CICC 21633 of sero-group IIa, CICC 21662,242-2, CICC 21632 of sero-group IIb, blood
CICC 21634, the CMCC 54002 of clear group IIc, the ATCC 19114 of the GIM 1.347 and sero-group L of sero-group IVb,
2006-1 and 3877-1.
Evaluation on specificity has used 20 plants of non-Listeria monocytogenes, respectively Ying Nuoke Listeria (Listeria altogether
Innocua), listeria ivanovii (Listeria ivanovii), Listera grayi (Listeria grayi), Lee Si Shi
This special bacterium (Listeria seeligeri), Wei Shi Listeria (Listeria welshimeri), Escherichia coli
(Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), Corynebacterium glutamicum
(Corynebacterium glutamicum), proteus mirabilis (Proteus mirabilis), Wei Demanshi bacillus
(Bacillus wiedmannii), thunder Allen Ginsberg Yokenella (Yokenella regensburgei), Granada vacation unit cell
Bacterium (Pseudomonas granadensis), Japan bacillus (Bacillus toyonensis), solution starch gemma bar
Bacterium (Paenibacillus amylolyticus), Staphylococcus sciuri (Staphylococcus sciuri), amber grape ball
Bacterium (Staphylococcus succinus), Xiamen Shewanella (Shewanella xiamenensis), Olso Moraxella
Bacterium (Moraxella osloensis), the solution huge coccus of casein (Macrococcus caseolyticus), format galactococcus
(Lactococcus garvieae)。
10 plants of Listeria monocytogenes and 20 plants of non-Listeria monocytogenes are inoculated into respectively in the LB liquid medium of 10mL,
It is put into shaken cultivation in shaking table, the revolving speed of shaking table is that 220rpm takes bacteria suspension 1mL respectively, obtain 30 after 37 DEG C of increasing bacterium 12h
Part bacteria suspension extracts genomic DNA using the DNA of bacteria extracts kit of TIANGEN Biotech (Beijing) Co., Ltd., obtains
30 parts of genomic DNAs.
30 parts of obtained genomic DNAs will be extracted takes 1 μ L to do PCR reaction respectively, and PCR reaction system and response procedures are strictly according to the facts
It applies described in example 1.PCR reaction product is subjected to electrophoresis, and judges the side of Listeria monocytogenes serotype as described in example 1 above
Method carrys out qualification result.PCR qualification result in embodiment 2 is as shown in table 2, Fig. 1 a and Fig. 1 b；Wherein, table 2 is PCR reaction system
The bacterial strain and its qualification result that Evaluation on specificity experiment uses, the M of Fig. 1 a and Fig. 1 b are represented as DL 1,000DNA Marker,
Swimming lane 1-10 is Listeria monocytogenes, and swimming lane 11-24 is non-Listeria monocytogenes, the corresponding strain of each swimming lane and strain name
It is shown in Table 2.The experimental results showed that PCR Serotype Identification method of the invention increases Liszt to 10 plants of the single of different serotypes
Bacteria strain all can correctly identify the sero-group belonging to it, accuracy with higher；To the bacterium of 20 plants of non-Listeria monocytogenes
Kind, the PCR method based on multi-target identification Listeria monocytogenes serotype that embodiment 2 provides all is accredited as feminine gender, therefore,
This method also specificity with higher.
Table 2
"-" indicates that qualification result is non-Listeria monocytogenes in table 2.
Above embodiments are only preferrred embodiment of the present invention, for explaining only the invention, are not intended to limit the present invention, this
Field technical staff should belong to guarantor of the invention without departing from change made under spirit of the invention, replacement, modification etc.
Protect range.
Sequence table
<110>South China Science & Engineering University
<120>a kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Listeria monocytogenes
<400> 1
acggtattac ttagcgatgt gc 22
<210> 2
<211> 23
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 2
caaactcaaa ataaacgctt caa 23
<210> 3
<211> 20
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 3
accgaccaat gtctacacgc 20
<210> 4
<211> 19
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 4
ttcacgcatt ctggcatct 19
<210> 5
<211> 21
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 5
gtggttctgg tcttgcctta g 21
<210> 6
<211> 22
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 6
gattgagaaa gagggttgaa aa 22
<210> 7
<211> 23
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 7
aaagcaatca gaaaatcaaa agg 23
<210> 8
<211> 24
<212> DNA
<213>Listeria Monocytogenes (Listeria monocytogenes)
<400> 8
gtacgatttt atacgttctt ccgc 24
Claims (8)
1. a kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype, which is characterized in that including such as
Lower step:
(1) Listeria monocytogenes strain inoculated to be measured is subjected to Zengjing Granule into culture medium, obtains enrichment culture medium；
(2) genomic DNA for using RNA isolation kit extraction step (1) described enrichment culture medium obtains DNA to be identified and extracts
Liquid；
(3) by step (2) the DNA extracting solution to be identified, it is described identification Listeria monocytogenes serotype PCR primer with
PCR reactant is uniformly mixed, and obtains PCR reaction system, is then carried out PCR reaction, is obtained PCR reaction product；
(4) step (3) the PCR reaction product is subjected to agarose gel electrophoresis, electrophoresis result figure is obtained, if the electrophoresis
Occurs 669bp and 500bp band in result figure, then the sero-group of determination step (1) the Listeria monocytogenes bacterial strain to be measured is
IIa, serotype are 1/2a or 3a；If only there is the band of 500bp, it is determined as sero-group IIb, i.e. serotype 1/2b, 3b
Or 7；If only there is the band of 669bp, 243bp and 500bp, it is determined as sero-group IIc, i.e. serotype 1/2c or 3c；If only
There is the band of 889bp and 500bp, is then determined as sero-group IVb, i.e. serotype 4b, 4d or 4e；If only occur 669bp,
The band of 889bp and 500bp is then determined as sero-group L, i.e. serotype 4a or 4c；If without going out on the electrophoresis result figure
The now above band does not contain Listeria monocytogenes then in step (1) sample to be identified, be judged as Listeria monocytogenes yin
Property.
2. the multiple PCR method according to claim 1 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, step (1) culture medium is TSB culture medium；The Zengjing Granule is the shaken cultivation in shaking table, the increasing bacterium
The temperature of culture is 36 ~ 38 DEG C, and the time of Zengjing Granule is 8 ~ 12 h, and the shaking speed of the Zengjing Granule is 210 ~ 230
rpm。
3. the multiple PCR method according to claim 1 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, the DNA concentration in step (2) the DNA extracting solution to be identified is 10-500 ng/ μ L.
4. the multiple PCR method according to claim 1 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, the PCR primer of step (3) the identification Listeria monocytogenes serotype includes PCR primer lmo2644, PCR primer
LMOf2365_0118, PCR primer lmo1119 and PCR primer lmo0733.
5. the multiple PCR method according to claim 4 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, the PCR primer lmo2644 includes forward primer lmo2644-F and reverse primer lmo2644-R；The forward direction
Primer lmo2644-F is as shown in SEQ ID NO:1, and the reverse primer lmo2644-R is as shown in SEQ ID NO:2；
The PCR primer LMOf2365_0118 includes forward primer LMOf2365_0118-F and reverse primer LMOf2365_
0118-R；The forward primer LMOf2365_0118-F is as shown in SEQ ID NO:3, the reverse primer LMOf2365_
0118-R is as shown in SEQ ID NO:4；
The PCR primer lmo1119 includes forward primer lmo1119-F and reverse primer lmo1119-R；The forward primer
Lmo1119-F is as shown in SEQ ID NO:5, and the reverse primer lmo1119-R is as shown in SEQ ID NO:6；
The PCR primer lmo0733 includes forward primer lmo0733-F and reverse primer lmo0733-R；The forward primer
Lmo0733-F is as shown in SEQ ID NO:7, and the reverse primer lmo0733-R is as shown in SEQ ID NO:8.
6. the multiple PCR method according to claim 1 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, step (3) the PCR reaction system includes based on volume parts:
5 parts of 10 × PCR reaction buffer；
1 part of dNTP solution；
1 part of PCR primer solution for identifying Listeria monocytogenes serotype；
1 part of DNA extracting solution to be identified；
1 part of Taq enzyme solution；
41 parts of water；
Wherein, the pH value of 10 × PCR reaction buffer is 8.2 ~ 8.4；The dNTP solution be dATP, dGTP, dTTP and
The solution that these four deoxyribonucleoside triphosphate equal proportions of dCTP are uniformly mixed with water, in the dNTP solution four kinds it is de-
The concentration of oxygen ribonucleotide triphosphate is 10 ~ 15 mM；The PCR primer solution for identifying Listeria monocytogenes serotype is
The solution that the PCR primer of identification Listeria monocytogenes serotype is uniformly mixed with water, the identification Listeria monocytogenes blood
The concentration of primer is each 8 ~ 12 μM in the PCR primer solution of clear type；The Taq enzyme solution is that Taq enzyme is uniformly mixed to obtain with water
Solution, the concentration of the Taq enzyme solution is 2 ~ 3 U/ μ L；The water is sterile and without nucleotide ultrapure water.
7. the multiple PCR method according to claim 1 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, the condition of step (3) the PCR reaction are as follows:
First in 95 ~ 98 DEG C of 2 ~ 4 min of initial denaturation, it is then extended circulation, finally extends 4 ~ 6 min under the conditions of 68 ~ 72 DEG C；
The Extended Cyclic are as follows: 14 ~ 16 s are denaturalized under the conditions of 95 ~ 98 DEG C, anneal 14 ~ 16 s under the conditions of 52 ~ 54 DEG C, and 68 ~ 72
Extend 29 ~ 31 s under the conditions of DEG C, carries out 30 ~ 35 circulations.
8. the multiple PCR method according to claim 1 based on specific target identification Listeria monocytogenes serotype,
It is characterized in that, the mass percent concentration of step (4) described Ago-Gel is 1.5-3wt%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110890129A (en) * | 2019-11-21 | 2020-03-17 | 华南理工大学 | Excavation method and application of Listeria monocytogenes serotype specific SNP |
| CN111118182A (en) * | 2019-12-30 | 2020-05-08 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of serotype listeria monocytogenes and rapid detection method thereof |
| CN112961926A (en) * | 2021-03-18 | 2021-06-15 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Primer, kit and method for simultaneously detecting Listeria monocytogenes CC87 and 88 strains |
| CN114944197A (en) * | 2022-05-18 | 2022-08-26 | 南开大学 | Automatic serotype analysis and identification method and system based on sequencing data |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060257894A1 (en) * | 2003-09-16 | 2006-11-16 | Michel Doumith | Molecular typing of listeria monocytogenes, hybridization supports and kits for said molecular typing |
| CN101029331A (en) * | 2007-01-30 | 2007-09-05 | 浙江大学 | Listeria and toxicity fast esting reagent kit |
| CN102286631A (en) * | 2011-09-14 | 2011-12-21 | 厦门出入境检验检疫局检验检疫技术中心 | Method for multiplex polymerase chain reaction (PCR) fast detection and five-Listeria-bacterium identification |
| CN103266179A (en) * | 2013-05-28 | 2013-08-28 | 上海交通大学 | Multiplex PCR (Polymerase Chain Reaction) detection method for salmonella typhimurium and serum variants of salmonella typhimurium |
| CN103602739A (en) * | 2013-11-13 | 2014-02-26 | 浙江省水产技术推广总站 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
| CN107746890A (en) * | 2017-11-29 | 2018-03-02 | 南京农业大学 | Identify the multiple PCR detection primer and method of Listeria monocytogenes serotype |
-
2019
- 2019-08-28 CN CN201910804288.0A patent/CN110423833B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060257894A1 (en) * | 2003-09-16 | 2006-11-16 | Michel Doumith | Molecular typing of listeria monocytogenes, hybridization supports and kits for said molecular typing |
| CN101029331A (en) * | 2007-01-30 | 2007-09-05 | 浙江大学 | Listeria and toxicity fast esting reagent kit |
| CN102286631A (en) * | 2011-09-14 | 2011-12-21 | 厦门出入境检验检疫局检验检疫技术中心 | Method for multiplex polymerase chain reaction (PCR) fast detection and five-Listeria-bacterium identification |
| CN103266179A (en) * | 2013-05-28 | 2013-08-28 | 上海交通大学 | Multiplex PCR (Polymerase Chain Reaction) detection method for salmonella typhimurium and serum variants of salmonella typhimurium |
| CN103602739A (en) * | 2013-11-13 | 2014-02-26 | 浙江省水产技术推广总站 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
| CN107746890A (en) * | 2017-11-29 | 2018-03-02 | 南京农业大学 | Identify the multiple PCR detection primer and method of Listeria monocytogenes serotype |
Non-Patent Citations (4)
| Title |
|---|
| ANNAËLLE KÉROUANTON等: "Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping", 《J MICROBIOL METHODS》 * |
| RAWOOL DB等: "A multiplex PCR for detection of Listeria monocytogenes and its lineages", 《J MICROBIOL METHODS》 * |
| 王毅等: "鉴定单增李斯特菌四个进化家系及两个亚系的多PCR方法的建立（英文）", 《中国人兽共患病学报》 * |
| 闫军等: "基于MALDI-TOF/TOF技术的单增李斯特菌四种血清型的快速鉴别", 《中国农业科技导报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110890129A (en) * | 2019-11-21 | 2020-03-17 | 华南理工大学 | Excavation method and application of Listeria monocytogenes serotype specific SNP |
| CN110890129B (en) * | 2019-11-21 | 2022-12-16 | 华南理工大学 | Excavation method and application of Listeria monocytogenes serotype specific SNP |
| CN111118182A (en) * | 2019-12-30 | 2020-05-08 | 广东省微生物研究所（广东省微生物分析检测中心） | Specific novel molecular target of serotype listeria monocytogenes and rapid detection method thereof |
| CN112961926A (en) * | 2021-03-18 | 2021-06-15 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Primer, kit and method for simultaneously detecting Listeria monocytogenes CC87 and 88 strains |
| CN112961926B (en) * | 2021-03-18 | 2024-01-09 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Primer, kit and method for simultaneously detecting listeria monocytogenes CC87 and listeria monocytogenes type 88 strains |
| CN114944197A (en) * | 2022-05-18 | 2022-08-26 | 南开大学 | Automatic serotype analysis and identification method and system based on sequencing data |
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