CN110423832A - A kind of PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160 - Google Patents
A kind of PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160 Download PDFInfo
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- CN110423832A CN110423832A CN201910804285.7A CN201910804285A CN110423832A CN 110423832 A CN110423832 A CN 110423832A CN 201910804285 A CN201910804285 A CN 201910804285A CN 110423832 A CN110423832 A CN 110423832A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention discloses a kind of PCR detection primers of Listeria monocytogenes based on specific gene target lmo0160.For the present invention for the specific detection target lmo0160 gene design specific primer of the Listeria monocytogenes newly excavated, forward primer sequence is 5 '-ACGATGGCAACACGGTAGGTC-3 ', reverse primer sequences 5 '-CGCCGTGGAATGCTTTTAT-3 '.The specific detection target is excavated by comparing genomics and bioinformatics and is obtained, and is selected from lmo0160 gene.The detection target has the characteristics that specificity is good and sensibility is high simultaneously.PCR detection primer provided by the invention has the characteristics that specificity is good, sensibility is high.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of single increasing Lee based on specific gene target lmo0160
The PCR detection primer of this special bacterium.
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.
Traditional method based on culture is currently main method (the visible standard GB/T of Listeria monocytogenes detection
4789.30-2016), although it is to the of less demanding of experimental facilities, 5~10 days are needed qualification cycle.Use immunological method
It is complicated for operation although identifying that qualification cycle is short, it is unfavorable for high-throughput detection, and the preparation of monoclonal antibody is more difficult.Benefit
Have the characteristics that with the detection that PCR method carries out Listeria monocytogenes easy, quick, sensitive.Currently, Listeria monocytogenes
The target that PCR detection uses is mainly virulence gene, such as hly, inlA, iap gene.CN 100463972 C,CN
The Chinese patents such as 101029331 B/patent application discloses the PCR primer of detection Listeria monocytogenes, these methods have behaviour
Make easy and quick feature, but there are still deficiencies in terms of sensibility and specificity.
Summary of the invention
In order to overcome deficiencies of the prior art, the object of the present invention is to provide one kind to be based on specific gene target
Mark the PCR detection primer of the Listeria monocytogenes of lmo0160.
The PCR detection primer of Listeria monocytogenes provided by the invention based on specific gene target lmo0160 is one
The PCR primer for the detection Listeria monocytogenes that species specificity is good, sensibility is high.
The purpose of the present invention is realized at least through one of following technical solution.
A kind of PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160, including forward direction draws
Object lmo0160-F and reverse primer lmo0160-R；The nucleotide sequence of the forward primer lmo0160-F such as SEQ ID NO:1
It is shown；The nucleotide sequence of the reverse primer lmo0160-R is as shown in SEQ ID NO:2.
The PCR detection primer of Listeria monocytogenes provided by the invention based on specific gene target lmo0160 can
It applies in detection Listeria monocytogenes.
The PCR detection primer of Listeria monocytogenes provided by the invention based on specific gene target lmo0160, it is corresponding
Specific target gene be lmo0160.
The PCR detection primer of Listeria monocytogenes provided by the invention based on specific gene target lmo0160 is being examined
The application in Listeria monocytogenes is surveyed, is included the following steps:
(1) sample to be tested is subjected to Zengjing Granule, the sample after obtaining Zengjing Granule；
(2) genomic DNA of the sample after extraction step (1) described Zengjing Granule, obtains the DNA of sample to be tested；
(3) using the DNA of step (2) described sample to be tested as template DNA, specific gene target is based on using described
The PCR detection primer of the Listeria monocytogenes of lmo0160 carries out PCR reaction, obtains PCR reaction product；
(4) step (3) the PCR reaction product is subjected to agarose gel electrophoresis, if occurring molecular weight in electrophoresis result
Size is the specific band of 402bp, then step (1) described sample to be tested contains Listeria monocytogenes, if not having in electrophoresis result
The specific band that molecular size range is 402bp is occurred, then increases Liszt without containing single in step (1) described sample to be tested
Bacterium.
Further, step (3) the template DNA concentration is 2.146fg/ μ L~2.146ng/ μ L.
Zengjing Granule described in step (1) of the present invention is the conventional steps in Listeria detection, and the purpose is to be enriched with
Listeria existing for trace in sample, method are recorded in standard GB/T 4789.30-2016, the specific steps are as follows: will
Sample inoculation increases bacterial context soup in Listeria, in 37 DEG C of 10~12h of shaken cultivation；Wherein the Listeria increases bacterial context soup
Ingredient are as follows: tryptone 17.0g/L, multivalent protein peptone 3.0g/L, yeast extract 6.0g/L, sodium chloride 5.0g/L, phosphoric acid hydrogen
Dipotassium 2.5g/L, glucose 2.5g/L, nalidixic acid 40mg/L and acriflavine 15mg/L.
Extraction described in step (2) of the present invention increases the genomic DNA of bacterium sample, and method is the Conventional bacteria of this field
Genome DNA extracting method, the specific steps are as follows: take 3mL bacterium solution in centrifuge tube, be centrifuged 1min, abandon supernatant, collect bacterium
Body；Add 800 μ L lysis buffers (40mM Tris- acetic acid, the 20mM sodium acetate of pH 7.8,1mM EDTA, 10%SDS), blows and beats
It mixes；After adding 400 μ L 5M NaCl to mix, it is centrifuged 12min；Supernatant is moved in another centrifuge tube, 10 μ L RNaseA are added
(10mg/mL) 37 DEG C of warm bath 30min；Isometric phenol/chloroform/isoamyl alcohol (25 ︰, 24 ︰ 1) extracting is added, mixes well extracting；From
Heart 8min collects supernatant, primary with chloroform, draws in supernatant to new centrifuge tube after being centrifuged 8min；Add 2 times of volumes anhydrous
It is centrifuged 8min after ethanol precipitation DNA, washs precipitating 2 times with the ethanol solution that concentration of volume percent is 70%.After drying, it is dissolved in
It is saved backup in 100 μ L TE in -20 DEG C.The bacterial genomes DNA extraction kit that commercialization can also be used, such as Tiangeng biochemistry
Bacterial genomes DNA extraction kit, the bacterial genomes DNA Rapid extraction kit of raw work biology, Solarbio it is thin
Bacterium genome DNA extracting reagent kit etc..
Pcr amplification reaction described in step (3), refers in PCR reaction system, in archaeal dna polymerase and the primer
To under the action of, by being recycled under the reaction condition successively through 95~98 DEG C of denaturation, 53~55 DEG C of annealing and 68~72 DEG C of extensions
Reaction 30~35 times, using Listeria monocytogenes genomic DNA as template DNA, using dNTP as substrate, in the template with institute
The specific fragment region for stating primer pair complementation carries out amplification accumulation.The PCR reaction system, comprising PCR reaction buffer,
Archaeal dna polymerase, dNTP, primer pair of the present invention and template DNA.It is 8 that wherein the PCR reaction buffer, which includes pH,
~9.5 0~20mM Tris HCl, 30~100mM KCl and 1~4mM MgCl2；The archaeal dna polymerase is Taq DNA
Polymerase, working concentration are 0.02~0.2U/ μ L；The dNTP is tetra- kinds of deoxyriboses of dATP, dGTP, dTTP and dCTP
The mixture of ribonucleoside triphosphote equal proportion composition, concentration are each 0.2~0.3mM；The template DNA concentration is 2.146fg/ μ L
~2.146ng/ μ L；The primer pair concentration is each 0.2~0.4 μM.PCR reaction system is in addition to primer sequence used in the present invention
Outside template DNA, remaining composition is conventional PCR method common prescription, is well known to those of ordinary skill in the art and grasp,
It can voluntarily be prepared by formula, the kit of commercialization can also be used.Currently, there are many for routine PCR reaction in the market
Kit is suitable for the method for the present invention, such as the Hot Start Taq 2X Master of New England Biolabs (NEB)
Mix, Taq 5X Master Mix etc., Takara'sPCR Amplification Kit etc., the KOD of TOYOBO
Taq DNA Polymerase, AmpliTaq DNA Polymerase of FX etc. and Invitrogen etc..It is of the present invention
PCR reaction condition be also routine PCR reaction condition, those of ordinary skill in the art know according to its conventional PCR method grasped
Know and archaeal dna polymerase type used can determine denaturation, annealing easily and extend time and temperature used；In addition, can also root
It is reacted according to condition provided by specification in commercial kit.
A kind of PCR detection primer of Listeria monocytogenes based on specific gene target lmo0160 provided by the invention,
The specific target gene that the PCR primer uses is lmo0160 (NCBI Gene ID:986846).
A kind of PCR detection primer of Listeria monocytogenes based on specific gene target lmo0160 provided by the invention
It is a pair of of specific primer pair, is made of upstream primer and downstream primer, sequence is respectively as follows:
Lmo0160-F:5 '-ACGATGGCAACACGGTAGGTC-3 ', as shown in SEQ ID NO:1；
Lmo0160-R:5 '-CGCCGTGGAATGCTTTTAT-3 ', as shown in SEQ ID NO:2.
The PCR detection primer of Listeria monocytogenes of the present invention based on specific gene target lmo0160 is to be based on
The Listeria monocytogenes PCR detection primer of specific detection target lmo0160 gene design.The lmo0160 gene coding
Albumen be peptide glycan binding protein.This gene all exists in the Listeria monocytogenes bacterial strain of known group, and known
It is all not present in the non-Listeria monocytogenes bacterial strain of genome, therefore can be used as the detection of Listeria monocytogenes.For described
Lmo0160 gene designs primer pair, detects Listeria monocytogenes for PCR method, draws with similar PCR in the prior art
Object is compared, and has higher specificity and sensibility.
The invention discloses a kind of specific detection target of Listeria monocytogenes and its corresponding PCR detection primers.
The present invention designs specific primer for the specific detection target lmo0160 gene of the Listeria monocytogenes newly excavated, just
It is 5 '-ACGATGGCAACACGGTAGGTC-3 ' (lmo0160-F), reverse primer sequences 5 '-to primer sequence
CGCCGTGGAATGCTTTTAT-3'(lmo0160-R).The specific detection target is believed by comparing genomics and biology
Breath is learned to excavate and be obtained, and lmo0160 gene is selected from.The detection target has the characteristics that specificity is good and sensibility is high simultaneously.This hair
Bright detection primer has the characteristics that specificity is good, sensibility is high.
Compared with prior art, the invention has the advantages that and the utility model has the advantages that
The PCR detection primer of Listeria monocytogenes provided by the invention based on specific gene target lmo0160；It is examined
Survey target lmo0160 has the characteristics that specificity is good and sensibility is high simultaneously；Therefore PCR detection primer provided by the invention can
Listeria monocytogenes are detected well.
Detailed description of the invention
Fig. 1 a is the PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160 in embodiment 2
The electrophoresis detection partial results figure of Evaluation on specificity；
Fig. 1 b is the PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160 in embodiment 2
Electrophoresis detection another part result figure of Evaluation on specificity.
Fig. 2 is the electrophoresis detection result of the sensitivity evaluation of Listeria monocytogenes PCR detection primer in embodiment 3.
Fig. 3 is the electrophoresis detection result of Listeria monocytogenes artificial contamination's product P CR detection in embodiment 4.
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.
In following embodiment, sensibility refers to being detected as positive sample number institute in the sample actually for the positive
The ratio accounted for, specificity refer to being detected as ratio shared by negative sample number in the sample actually for feminine gender.
Embodiment 1
Excavate Listeria monocytogenes PCR detection primer
From the reference genome EGD-e (NC_003210.1) of NCBI downloading Listeria monocytogenes, it is divided into 1000bp
These segments are compared with 202 Listeria monocytogenes full-length genomes using BLASTN, comparison are obtained by the segment of size
LM kind in conserved sequence be compared again with the genome of 114 listeria other species, what will finally be obtained single increases
Listeria specific sequence is compared using the sequence for belonging to Bacteria branch in online BLAST tool and NT database
It is right, with further determine that its specificity, finally obtain Listeria monocytogenes specificity target lmo0160 (NCBI Gene ID:
986846).Design of primers is carried out using 6 couples of specificity target lmo0160 of software Primer Premier, determines primer sequence
It is as follows:
Lmo0160-F:5 '-ACGATGGCAACACGGTAGGTC-3 ', as shown in SEQ ID NO:1；
Lmo0160-R:5 '-CGCCGTGGAATGCTTTTAT-3 ', as shown in SEQ ID NO:2.
In order to verify the accuracy of detection primer of the present invention, using online tool Primer-BLAST by the primer of design with
The sequence for belonging to Bacteria branch in the NT database of NCBI is compared, and obtains the column of the amplified production on target sequence
Table.If containing the genome of a certain bacterial strain in amplified production list, then it is assumed that the testing result to the bacterial strain is the positive；If amplification
The genome of a certain bacterial strain is not contained in the list of product, then it is assumed that the testing result to the bacterial strain is feminine gender.PCR of the invention
Detection primer correctly will test out whole 217 genomes in 217 Listeria monocytogenes genomes of NT database
Corresponding bacterial strain test positive, and be not the positive by the genome detection of non-Listeria monocytogenes.To in existing patent
Detection primer carries out identical verifying, though the primer specificity in the inventions such as 104342496 B of CN is higher, only correctly examines
196 Listeria monocytogenes genomes in database are measured, sensibility susceptibility is not high enough；The invention such as 102676647 B of CN
Although primer sensibility susceptibility in is sufficiently high, is the positive, specificity by the genome detection of 4 non-Listeria monocytogenes
Not high enough, as shown in table 1, table 1 is that the PCR detection of the Listeria monocytogenes based on specific gene target lmo0160 is drawn
The sensibility and specificity of object evaluates tables of data.Therefore the Listeria monocytogenes that the present invention excavates detect target and according to these
The detection primer for detecting drone design compared to existing patent, while having the characteristics that sensibility susceptibility height and specificity are high.
Table 1
Using the above-described primer pair (PCR of the Listeria monocytogenes based on specific gene target lmo0160
Detection primer), it can establish the PCR detection method of Listeria monocytogenes.
The PCR reaction system total volume is 50 μ L, including 10 × PCR reaction buffer, 5 μ L, template DNA 1 μ L, remaining
Person is sterile DEPC water (ultrapure water sterile and without nucleotide)；Wherein contain dNTP 0.25mM in PCR reaction system, draws
Lmo0160-F0.2 μM of object, 0.2 μM of lmo0160-R and Taq enzyme 2.5U.
Affiliated PCR response procedures are as follows: 95 DEG C of initial denaturation 3min start amplification cycles later, and the program of each circulation is,
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 testing result: take the PCR reaction product of 5 μ L in the Ago-Gel that mass percent concentration is 2wt%
Middle carry out electrophoresis is judged as the Listeria monocytogenes positive if there is the band of 402bp, if not occurring the band of 402bp,
It is judged as Listeria monocytogenes feminine gender.
Embodiment 2
The specificity of the PCR detection primer of Listeria monocytogenes based on specific gene target lmo0160 of the invention
Evaluation
10 plants of single increasing Liszts have been used altogether to the Evaluation on specificity of Listeria monocytogenes PCR detection primer of the invention
Bacterium, respectively CICC 21633, CICC 21662,242-2, CICC 21632, CICC 21634, CMCC 54002, GIM
1.347、ATCC 19114、2006-1、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 after 37 DEG C of increasing bacterium 12h, raw using Tiangeng
The DNA of bacteria extracts kit for changing scientific and technological (Beijing) Co., Ltd extracts genomic DNA.
Obtained genomic DNA, which will be extracted, takes 1 μ L to do in PCR reaction, PCR reaction system and response procedures such as embodiment 1
It is described.The PCR reaction product is gone to carry out electrophoresis, and the method for the detection Listeria monocytogenes as provided in embodiment 1 judges to tie
Fruit.PCR testing result is as shown in table 2, Fig. 1 a and Fig. 1 b.Fig. 1 a and Fig. 1 b are that specific gene target is based in embodiment 2
The electrophoresis detection result figure of the Evaluation on specificity of the PCR detection primer of the Listeria monocytogenes of lmo0160, in Fig. 1 a and Fig. 1 b
M is expressed as DL 1,000DNA Marker；Swimming lane 1-10 is Listeria monocytogenes, and swimming lane 11-24 is non-Listeria monocytogenes, tool
Body strain and strain name are shown in Table 1.
The experimental results showed that (table 2, Fig. 1 a and Fig. 1 b), provided by the invention based on specific gene target lmo0160's
The PCR detection primer of Listeria monocytogenes can all correctly detect as the positive, tool 10 plants of different Listeria monocytogenes bacterial strains
There is higher sensibility；It is provided by the invention to be based on specific gene target to the strain of 20 plants of non-Listeria monocytogenes
The PCR detection primer of the Listeria monocytogenes of lmo0160 is all detected as feminine gender, therefore, provided by the invention based on specific base
Because of the PCR detection primer also specificity with higher of the Listeria monocytogenes of target lmo0160.
Table 2
In table 2+indicate that testing result is the positive；Indicate that testing result is feminine gender.
Embodiment 3
The inspection of the PCR detection primer of Listeria monocytogenes provided by the invention based on specific gene target lmo0160
Survey limit measurement
The genomic DNA of Listeria monocytogenes GIM 1.347 is dense using ultramicron ultraviolet specrophotometer measurement DNA
Degree, measuring initial concentration is 107.3ng/ μ L.Continuous 10 times of gradient dilutions are carried out to genomic DNA using sterile water, are obtained dense
Degree is the genomic DNA of 107.3ng/ μ L-10.7fg/ μ L, and the DNA of each dilution gradient takes 1 μ L that PCR reaction is added as template
System (50 μ L).Amplification is as shown in table 3 and attached drawing 2；Table 3 is that PCR reaction system limits result to the detection of genomic DNA
Table, the M in Fig. 2 are expressed as DL 1,000DNA Marker, and DNA concentration is respectively in the corresponding PCR system of swimming lane 1-8
2.146ng/μL、214.6pg/μL、21.46pg/μL、2.146pg/μL、214.6fg/μL、21.46fg/μL、2.146fg/μL、
0.2146fg/μL.When the concentration of Listeria monocytogenes genomic DNA is between 2.146ng/ μ L~2.146fg/ μ L, PCR is anti-
Answer system that can amplify positive findings, when DNA concentration drops to 0.2146fg/ μ L, reaction system can not amplify band,
It is determined as feminine gender.Therefore, which is limited to 2.146fg/ μ L to the detection of Listeria monocytogenes genomic DNA.Therefore, originally
The PCR detection primer for inventing the Listeria monocytogenes based on specific gene target lmo0160 provided increases Liszt for single
The detection of bacterium genomic DNA sensitivity with higher.
Table 3
In table 3+indicate that testing result is the positive；Indicate that testing result is feminine gender.
Embodiment 4
The PCR of Listeria monocytogenes artificial contamination's food is detected
By Listeria monocytogenes colony inoculation in TSB culture medium shaken cultivation, 1mL bacterium solution is taken to carry out 10 times with physiological saline
Gradient dilution, the dilution bacterium solution of every 10mL milk sample access 1mL, at the beginning of the Listeria monocytogenes in the milk sample of artificial contamination
Beginning concentration is measured using colony counting method, and the initial concentration for measuring bacterium solution is 8.3 × 104CFU/10mL milk, 8.3 × 103CFU/
10mL milk, 8.3 × 102CFU/10mL milk, 8.3 × 101CFU/10mL milk and 8.3 × 100CFU/10mL milk.It will connect
The Listeria that milk sample (10mL) after kind is separately added into 90mL increases in bacterial context soup, shaken cultivation at 37 DEG C.In 4h, 6h,
8h, 10h, 12h sample 1mL, extract genome using the DNA of bacteria extracts kit of TIANGEN Biotech (Beijing) Co., Ltd.
DNA.PCR reaction as described in Example 1 is carried out as template to genomic DNA using what is extracted.It is different for different vaccination amount
Increase the PCR testing result of bacterium time as shown in table 4 and attached drawing 3；Table 4 is the PCR of Listeria monocytogenes artificial contamination milk sample
Testing result table, A, B, C, D, E in Fig. 3 are respectively initial inoculum 8.3 × 104CFU/10mL、8.3×103CFU/10mL、
8.3×102CFU/10mL、8.3×101CFU/10mL、8.3×100The PCR testing result figure of the milk sample of CFU/10mL；Figure
M in 3 is expressed as DL 1, and 000DNA Marker, swimming lane 1-6 are respectively that the PCR after increasing bacterium 0h, 4h, 6h, 8h, 10h, 12h is produced
Object.After increasing bacterium 10h, initial inoculum is that the Listeria monocytogenes of 8.3CFU can be by PCR body in the milk sample of artificial contamination
It is test positive.That is, this reaction system is limited to 8.3CFU/10mL milk to the detection of Listeria monocytogenes in milk sample.
Therefore, the inspection established using the PCR detection primer of the Listeria monocytogenes of the invention based on specific gene target lmo0160
The survey method Listeria monocytogenes micro for milk sample also have a preferable detectability, and with conventional method 5-10 days
Qualification cycle is compared, and the present invention only needs 10h that can effectively detect Listeria monocytogenes micro in food, when shortening detection
Between.
Table 4
In table 4+indicate that testing result is the positive；Indicate that testing result is feminine gender.
In conclusion the PCR of the Listeria monocytogenes provided by the invention based on specific gene target lmo0160 is detected
Primer is higher for the detection specificity and sensibility of Listeria monocytogenes, and in Listeria monocytogenes nucleic acid and food
The detection sensitivity of Listeria monocytogenes is higher, there is more comprehensive detectability.
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 PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>Listeria monocytogenes (Listeria Monocytogenes)
<400> 1
acgatggcaa cacggtaggt c 21
<210> 2
<211> 19
<212> DNA
<213>Listeria monocytogenes (Listeria Monocytogenes)
<400> 2
cgccgtggaa tgcttttat 19
Claims (4)
1. a kind of PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160, which is characterized in that packet
Include forward primer lmo0160-F and reverse primer lmo0160-R；The nucleotide sequence such as SEQ of the forward primer lmo0160-F
Shown in ID NO:1；The nucleotide sequence of the reverse primer lmo0160-R is as shown in SEQ ID NO:2.
2. the PCR detection primer of the Listeria monocytogenes described in claim 1 based on specific gene target lmo0160 is being examined
Survey the application in Listeria monocytogenes.
3. the PCR detection primer of the Listeria monocytogenes as claimed in claim 2 based on specific gene target lmo0160 is being examined
Survey the application in Listeria monocytogenes, which comprises the steps of:
(1) sample to be tested is subjected to Zengjing Granule, the sample after obtaining Zengjing Granule；
(2) genomic DNA of the sample after extraction step (1) described Zengjing Granule, obtains the DNA of sample to be tested；
(3) using the DNA of step (2) described sample to be tested as template DNA, specific gene target lmo0160 is based on using described
Listeria monocytogenes PCR detection primer carry out PCR reaction, obtain PCR reaction product；
(4) step (3) the PCR reaction product is subjected to agarose gel electrophoresis, if occurring molecular size range in electrophoresis result
For the specific band of 402bp, then step (1) described sample to be tested contains Listeria monocytogenes, if without going out in electrophoresis result
The specific band that existing molecular size range is 402bp does not contain Listeria monocytogenes then in step (1) described sample to be tested.
4. the PCR detection primer of the Listeria monocytogenes according to claim 3 based on specific gene target lmo0160
Detection Listeria monocytogenes in application, which is characterized in that step (3) the template DNA concentration be 2.146 fg/ μ L ~
2.146 ng/μL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910804285.7A CN110423832A (en) | 2019-08-28 | 2019-08-28 | A kind of PCR detection primer of the Listeria monocytogenes based on specific gene target lmo0160 |
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