CN103602739A - Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit - Google Patents
Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit Download PDFInfo
- Publication number
- CN103602739A CN103602739A CN201310571311.9A CN201310571311A CN103602739A CN 103602739 A CN103602739 A CN 103602739A CN 201310571311 A CN201310571311 A CN 201310571311A CN 103602739 A CN103602739 A CN 103602739A
- Authority
- CN
- China
- Prior art keywords
- cryopreservation tube
- listeria monocytogenes
- checked
- positive control
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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]
-
- 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
- C12Q2531/00—Reactions of nucleic acids characterised by
- C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
- C12Q2531/113—PCR
-
- 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
- C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
- C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
- C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quick typing kit for major serotypes of listeria monocytogenes. The kit comprises freezing tubes and a PCR (Polymerase Chain Reaction) tube, wherein the freezing tubes include a freezing tube A, a freezing tube B, a freezing tube C, a freezing tube D, a freezing tube E, a freezing tube F and a freezing tube G; the freezing tube A is filled with a freeze-dried powder prepared from a mixed solution of Taq DNA (deoxyribonucleic acid) polymerase, 10*buffer, dNTP (diethyl-nitrophenyl thiophosphate) mixture and a primer; the freezing tube B is filled with a lysate; each of the freezing tubes C-F is filled with a positive reference buffer solution for one of the major serotypes of the listeria monocytogenes; the freezing tube G is filled with a diluent. The kit disclosed by the invention is capable of distinguishing total 4 major serotypes of the listeria monocytogenes accurately and quickly through one step of PCR; the method is low in price, simple and convenient to operate, and good in specificity and sensitivity; the sensitivity is capable of reaching 10<2>CFU/mL; the method is free of cross reaction, and accurate and reliable in results, and the results are simple and easy to read; furthermore, the quick typing kit is applicable for large-flux typing operation.
Description
(1) technical field
The present invention relates to a kind of differential diagnosis of bacterial pathogen serotype, based on multiple PCR method, set up the fast typing technology of identifying the main serotype of Listeria monocytogenes, i.e. Listeria monocytogenes main serotype fast typing test kit and application method.
(2) background technology
Listeria monocytogenes (Listeria monocytogenes) is that important people beast fish suffers from foodborne bacterial pathogens altogether, strong to environmental resistance, can be in higher salt concn (10%NaCl) and wide in range pH(pH4.5～9) and temperature range (0～45 ℃) in growth, and can form pod membrane, thereby Listeria monocytogenes is extensively present in nature (comprising soil, water source, plant and animal body etc.), easily pollute various food (meat, milk, sea-food and vegetables etc.).Listeria monocytogenes can cause gastro-enteritis, septicemia, meningitis and miscarriage etc., its lethality rate (20～30%) is far above other common foodborne bacterial pathogenses, as Salmonella enteritidis (approximately 0.38%), Campylobacter (0.02～0.1%), vibrios (0.005～0.1%) etc.Within 2000, by WHO food safety work program, classified as one of food-borne pathogens of detection, its harm seriousness has some idea of.
According to the serological reaction of thalline/flagellar antigen (O/H), Listeria monocytogenes can be divided into 13 serotypes, i.e. 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7 types.Wherein serotype 1/2a, 1/2b, 1/2c and 4b are main serotype, account for food and the more than 99% of patient's strain isolated; Other serotypes separation rate are extremely low.Be not that all serotype all shows identical virulence.Serotype 1/2a separation rate in food is the highest, but most people's invasion and attack type listeriosis cases of breaking out or distributing are caused by 4b type, and gastro-enteritis type listeriosis case is many, by 1/2a type and 1/2b type, caused, other serotypes rarely have the case that causes that people falls ill.Aggressive listeriosis and gastro-enteritis listeriosis mainly contain following difference: the contaminated food products that causes gastro-enteritis is taken content of molds (pathogen load) greatly, morbidity anxious (18～27h), symptom comprises heating, diarrhoea, vomiting, arthrodynia and headache etc., and the infected is mostly healthy population; And aggressive case mainly causes hypoimmunity crowd's lethality symptom.Meningitis patient's 4b type separation rate is significantly higher than general patient, and 4b type the infected's mortality ratio (26%) is apparently higher than other serotype the infected (16%), points out 4b type may have even better virulence.Therefore, need the method for rapid typification that foundation can be differentiated each main serotype of Listeria monocytogenes badly.
At present, the Serotypes of Listeria monocytogenes is mainly serological method, depends on commercial thalline and flagellar antigen, not only expensive, and while there is operational cost, multiple, the test-results of effort, cross reaction is difficult for the drawbacks such as interpretation, is not especially suitable for the somatotype operation of large flux.By comparative genomics method, filter out successively the special gene locus of serotype, but there is no so far the method for rapid typification of the main serotype of Listeria monocytogenes both at home and abroad.The present invention will be by the optimization to the analysis of serotype specific gene, primer and testing conditions, designs a kind of accurate, sensitive, easy, multiple PCR detection kit fast, to filling up above-mentioned blank.
(3) summary of the invention
The object of the invention is to provide a kind of accurate, sensitive, easy, the main Serotypes test kit of Listeria monocytogenes and using method fast.
The technical solution used in the present invention is:
The invention provides the main serotype fast typing of a kind of Listeria monocytogenes test kit, described test kit comprises cryopreservation tube and PCR reaction tubes, described cryopreservation tube comprises cryopreservation tube A, cryopreservation tube B, cryopreservation tube C, cryopreservation tube D, cryopreservation tube E, cryopreservation tube F and cryopreservation tube G, described cryopreservation tube A is equipped with the Taq archaeal dna polymerase for PCR reaction, 10 * buffer, lyophilized powder prepared by the mixed solution of dNTP mixture and primer, cryopreservation tube B is equipped with lysate, cryopreservation tube C, cryopreservation tube D, cryopreservation tube E, cryopreservation tube F is equipped with respectively Listeria monocytogenes 1/2a type positive control damping fluid, Listeria monocytogenes 1/2b type positive control damping fluid, Listeria monocytogenes 1/2c type positive control damping fluid, Listeria monocytogenes 4b type positive control damping fluid, cryopreservation tube G is equipped with diluent, described diluent is autoclaving distilled water, described positive control damping fluid be Listeria monocytogenes EGD-e(serotype 1/2a), Listeria monocytogenes 54004(serotype 1/2b), Listeria monocytogenes 54002(serotype 1/2c), Listeria monocytogenes ATCC19115(serotype 4b) be seeded in respectively in BHI substratum, after 37 ℃ of overnight incubation, get 100 ℃ of bacterium liquid and boil centrifugal abandoning supernatant after deactivation, get precipitation resuspended with distilled water, make positive control buffer sample,
In described cryopreservation tube A, the sequence of primer is:
L1-F：AGCAAAATGCCAAAACTCGT
L1-R：CATCACTAAAGCCTCCCATTG
L2-F：AGTGGACAATTGATTGGTGAA
L2-R：CATCCATCCCTTACTTTGGAC
L3-F：AGGGCTTCAAGGACTTACCC
L3-R：ACGATTTCTGCTTGCCATTC
L4-F：AGGGGTCTTAAATCCTGGAA
And L4-R:CGGCTTGTTCGGCATACTTA.
Further, described PCR reaction tubes is fixed according to detected sample quantity, and in test kit, described PCR reaction tubes is 100～300 conventionally.
Further, lyophilized powder in preferred described cryopreservation tube A is first at-80 ℃ of Ultralow Temperature Freezer pre-freeze 10h by mixed solution, put into Freeze Drying Equipment (loft drier pressure 0.03MPa), temperature is made as-40 ℃ (sublimation drying stages) lasting freeze-drying 30h, removes 90% above moisture again; Temperature rises to 25 ℃ to carry out parsing-desiccation 2h(temperature rise rate is 1.5 ℃/min afterwards), make moisture content reach 5%(mass concentration) below, obtain lyophilized powder; Described every 8 μ L mixed solutions consist of: Taq archaeal dna polymerase 0.4 μ L, 10 * buffer3 μ L, dNTP mixture(is containing dATP, dCTP, each 10mM of dGTP, dTTP) 0.6 μ L, concentration is each 0.5 μ L of L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R primer of 50 μ M, and dATP, dCTP, dGTP and dTTP that described dNTP mixture is 10mM by final concentration form.
Further, in described cryopreservation tube B, lysate final concentration consists of: volumetric concentration 4%Triton-X100,5mg/mL NaN
3, 1mol/L Tris, pH is that 8.0(regulates with 10M aqueous sodium hydroxide solution), solvent is distilled water, compound method: Triton-X1004mL, NaN
3(final concentration 5mg/mL) 500mg, Tris(final concentration 1mol/L) 12.114g, with 10M sodium hydroxide, adjust pH to 8.0, distilled water is settled to 100mL.
Further, preferably each test kit has 100 PCR reaction tubess, in described cryopreservation tube A the amount of lyophilized powder with freeze-drying before the volume of mixed solution count the consumption that 800 μ L(meet 100 PCR reaction tubess), in described cryopreservation tube B, lysate 1mL is housed, cryopreservation tube C～cryopreservation tube F is equipped with positive control damping fluid 0.5mL separately, cryopreservation tube G is equipped with 1.5mL diluent, and (diluent in cryopreservation tube G is the disposable cryopreservation tube A that adds conventionally, for 100 PCR reactions), in every cryopreservation tube, sample-loading amount is fixed according to PCR pipe number, in cryopreservation tube A the amount of lyophilized powder in freeze-drying before mixeding liquid volume, in mixeding liquid volume and cryopreservation tube G, diluent volume is than being 8:15, the suspension 15 μ L that each PCR reaction needed diluent is prepared with lyophilized powder suspendible, lysate 10 μ L in cryopreservation tube B, positive control damping fluid 5 μ L or sample to be checked 5 μ L.
The present invention also provides a kind of method of utilizing the main serotype fast typing of described Listeria monocytogenes test kit to carry out the main Serotypes of Listeria monocytogenes, described classifying method carries out as follows: (1) adds to cryopreservation tube A by diluent in cryopreservation tube G, resuspended, mix and make suspension, deposit in-20 ℃ standby (PCR reaction before first at room temperature thaws); Described lyophilized powder consumption in freeze-drying before mixeding liquid volume, in described cryopreservation tube G, diluent and mixeding liquid volume are than being 15:8;
(2) lysate in cryopreservation tube B is added respectively to PCR reaction tubes, be divided into contrast PCR reaction tubes and PCR reaction tubes to be checked, successively the positive control damping fluid in cryopreservation tube C, cryopreservation tube D, cryopreservation tube E and cryopreservation tube F is added in corresponding contrast PCR reaction tubes again, sample to be checked is added to PCR reaction tubes to be checked, mix, and then add the suspension in step (1) cryopreservation tube A in each PCR reaction tubes, carry out immediately pcr amplification; Described sample to be checked is that Listeria monocytogenes is seeded in BHI substratum, after 37 ℃ of overnight incubation, gets 100 ℃ of bacterium liquid and boils centrifugal abandoning supernatant after deactivation, gets precipitation resuspended with autoclaving distilled water, makes sample to be checked; In described cryopreservation tube B, in lysate and cryopreservation tube A, the volume ratio of suspension is 2:3, the volume ratio of the positive control damping fluid in described cryopreservation tube B in lysate and cryopreservation tube C, cryopreservation tube D, cryopreservation tube E and cryopreservation tube F is 2:1, and in described cryopreservation tube B, lysate is 2:1 with sample volume ratio to be checked; Conventionally in PCR reaction tubes, 30 μ L reaction solutions are housed, i.e. lysate 10 μ L in the 15 μ L of suspension in cryopreservation tube A, cryopreservation tube B, contrast liquid or sample liquid 5 μ L; Described BHI substratum is joined method by BHI medium standard, and 3.7gBHI powder is dissolved in 100mL distilled water, autoclaving;
(3) each PCR reaction tubes of step (2) is increased on PCR instrument by following reaction conditions: 95 ℃ of preheating 3min; 95 ℃ of sex change 45s, 55 ℃ of annealing 30s, 72 ℃ are extended 55s, 25 circulations; 72 ℃ keep 5min;
(4) by the electrophoresis in mass concentration 1% sepharose and 0.5 * tbe buffer liquid respectively of the product after step (3) amplification, use gel imaging system reading of data record after Goldview dyeing;
(5) result is judged: after detection, what the positive control solution in sample to be checked and cryopreservation tube C amplified 691bp band simultaneously is the Listeria monocytogenes 1/2a type positive; What in sample to be checked and cryopreservation tube D, positive control solution amplified 471bp band simultaneously is the Listeria monocytogenes 1/2b type positive; What in sample to be checked and cryopreservation tube E, positive control solution amplified 691bp, two bands of 906bp simultaneously is the Listeria monocytogenes 1/2c type positive; What in sample to be checked and cryopreservation tube F, positive control solution amplified 471bp, two bands of 597bp simultaneously is the Listeria monocytogenes 4b type positive; Positive control solution there is band and sample to be checked without band for the main serotype of non-Listeria monocytogenes; All without band or sample to be checked, there is band and positive control solution need again detect and judge without band in sample to be checked and positive control solution.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the invention provides the main serotype fast typing of a kind of Listeria monocytogenes test kit, this test kit only step PCR reaction just can be differentiated the whole 4 kinds of main serotypes of Listeria monocytogenes accurately and rapidly.Compare with the traditional Serotypes method based on agglutination reaction of serum, the method is cheap, easy and simple to handle, and specificity and susceptibility are good, and susceptibility can reach 10
2cFU/mL, no cross reaction, result is also simple readability accurately and reliably, is applicable to the somatotype operation of large flux.
(4) accompanying drawing explanation
Fig. 1 is gel electrophoresis picture,
In A, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 1/2a positive reference substance, and swimming lane 2 is sample to be checked (1/2a type is positive).
In B, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 1/2b positive reference substance, and swimming lane 2 is sample to be checked (1/2b type is positive).
In C, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 1/2c positive reference substance, and swimming lane 2 is sample to be checked (1/2c type is positive).
In D, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 4b positive reference substance, and swimming lane 2 is sample to be checked (4b type is positive).
In E, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 1/2a positive reference substance, and swimming lane 2 is sample to be checked (feminine gender).
In F, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 1/2b positive reference substance, and swimming lane 2 is sample to be checked (feminine gender).
In G, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 1/2c positive reference substance, and swimming lane 2 is sample to be checked (feminine gender).
In H, swimming lane M is 2000bp DNA Ladder Marker, and swimming lane 1 is serotype 4b positive reference substance, and swimming lane 2 is sample to be checked (feminine gender).
In I, swimming lane M is 2000bp DNA Ladder Marker, the positive reference substance of swimming lane 1 (having no band), and swimming lane 2 is sample to be checked (feminine gender).
In J, swimming lane M is 2000bp DNA Ladder Marker, the positive reference substance of swimming lane 1 (having no band), and swimming lane 2 is sample to be checked (positive).
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The main serotype fast typing of embodiment 1 Listeria monocytogenes test kit
One, cryopreservation tube forms
A pipe is lyophilized powder, lyophilized powder content in freeze-drying before mixeding liquid volume, mixed solution for 100 PCR reactions is 800 μ L, every 8 μ L mixed solutions are commercial by Taq DNA polymerase() 0.4 μ L, 10 * buffer(is commercial) 3 μ L, dNTP mixture0.6 μ L(is commercial, dATP, dCTP, dGTP and dTTP final concentration are 10mM), primer (Shanghai Ying Jun Bioisystech Co., Ltd is synthetic) L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, each 0.5 each primer concentration of μ L(of L4-F, L4-R are 50 μ M) form; The preparation of lyophilized powder be first by mixed solution at-80 ℃ of Ultralow Temperature Freezer pre-freeze 10h; Put into Freeze Drying Equipment (loft drier pressure 0.03MPa), temperature is made as-40 ℃ (sublimation drying stages) lasting freeze-drying 30h, removes 90% above moisture again; Temperature rises to 25 ℃ to carry out parsing-desiccation 2h(temperature rise rate is 1.5 ℃/min afterwards), moisture content is reached below 5%, make lyophilized powder, be stored in-20 ℃.
B pipe is equipped with 1mL lysate, compound method: Triton-X1004mL, NaN
3(final concentration 5mg/mL) 500mg, Tris(final concentration 1mol/L) 12.114g, with 10M aqueous sodium hydroxide solution, adjust pH to 8.0, distilled water is settled to 100mL, is stored in-20 ℃.
C-F pipe is equipped with 0.5mL solution separately, is 4 kinds of positive reference substances (Listeria monocytogenes 1/2a type, 1/2b type, 1/2c type and 4b type positive control damping fluids), is stored in-20 ℃.
G pipe contains 1.5mL solution, for diluent (being autoclaving distilled water), is stored in 4 ℃.
Primer sequence:
L1-F：AGCAAAATGCCAAAACTCGT
L1-R：CATCACTAAAGCCTCCCATTG
L2-F：AGTGGACAATTGATTGGTGAA
L2-R：CATCCATCCCTTACTTTGGAC
L3-F：AGGGCTTCAAGGACTTACCC
L3-R：ACGATTTCTGCTTGCCATTC
L4-F：AGGGGTCTTAAATCCTGGAA
L4-R：CGGCTTGTTCGGCATACTTA
100 of PCR reaction tubess.
Two, application of sample and detecting step explanation
(1) 1.5mL diluent in cryopreservation tube G being added to mixed solution freeze-drying front volume in cryopreservation tube A(cryopreservation tube A is 800 μ L), resuspended, mix and make suspension, deposit in-20 ℃ standby;
(2) the 10 μ L of the lysate in cryopreservation tube B are added respectively to 5 (every pipe 10 μ L lysates) PCR reaction tubess, be divided into contrast PCR reaction tubes C-F and PCR reaction tubes to be checked, again the 5 μ L of the positive control solution in cryopreservation tube C-F are added respectively in contrast PCR reaction tubes C-F, sample 5 μ L to be checked are added in PCR reaction tubes to be checked, mix, then the suspension 15 μ L in step (1) cryopreservation tube A are added respectively to above-mentioned contrast PCR reaction tubes C-F and PCR reaction tubes to be checked, carry out immediately pcr amplification;
(3) PCR reaction tubes is increased on PCR instrument (Life Technologies, Veriti) by following reaction conditions: 95 ℃ of 3min; 95 ℃ of 45s, 55 ℃ of 30s, 72 ℃ of 55s, 25 circulations; 72 ℃ of 5min;
(4), by the product electrophoresis in mass concentration 1% sepharose, 0.5 * tbe buffer liquid after amplification, after Goldview dyeing (commercial), use gel imaging system (sky, Shanghai energy 4500SF) reading of data analytical results.
Below by test, result of use of the present invention is proved and described.
1 materials and methods
1.1 bacterial strains and cultivation
134 strain Listeria monocytogenes (separated source is in Table 1) are carried out to enlarged culturing, through selective coloration culture medium (CHROM agar, France's Kerma (unit of kinetic energy) is good) with Vitek(France Mei Liai) method carries out bacterial classification confirmation, be Listeria monocytogenes, for ease of experiment, each bacterial strain except Reference Strains is numbered to (refer to table 1, M4 to SH4 is capable, M1 to SH3 is capable, M9 to NB28 is capable, M5 to 125SL1 is capable, M7 to W1-111 is capable).With the gram-positive microorganism (G that Listeria monocytogenes sibship is near, G+C content is low
+ low) as listera innocua (Listeria innocua), listeria ivanovii (Listeria ivanovii), Wei Shi listeria bacteria (Listeria welshimeri), Sai Shi listeria bacteria (Listeria seeligeri), listeria grayi (Listeria grayi), subtilis (Bacillus subtilis), bacillus cereus (Bacillus cereus), enterococcus faecalis (Enterococcus faecalis), streptococcus aureus (Staphylococcus aureus), swine streptococcus (Streptococcus suis), streptococcus equi epizootic disease subspecies (Streptococcus.equi subsp.zooepidemicus), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus curvatus (Lactobacillus crispatus) and other 12 kinds of negative control bacterial strain Aeromonas hydrophilas (Aeromonas hydrophila), Pseudomonas aeruginosa (Pseudomonas aeruginosa), vibrio alginolyticus (Vibrio alginolyticus), Vibrio parahaemolyticus (Vibrio parahaemolyticus), colon bacillus (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), Salmonella enteritidis (Salmonella enteritidis), white dysentery Salmonellas (Salmonella pullorum), shigella flexneri (Shigella flexneri), actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), pasteurella multocida (Pasteurella multocida), segmental bronchus sepsis is rich also preserves (referring to table 1) by this laboratory for Salmonella (Bordetella bronchiseptica).Each inoculation is in 5mL BHI (DIFCO) substratum (Oxoid, Hampshire, England), and 37 ℃ of shaking culture are spent the night.
1.2 serotypes based on serological method
Through based on thalline/flagellar antigen (O/H) and sero-fast agglutination reaction, all Listeria monocytogenes bacterial strains are carried out to traditional Serotypes.In 134 strain Listeria monocytogenes, contain 50 strains of 1/2a type bacterial strain, 29 strains of 1/2b type bacterial strain, 17 strains of 1/2c type bacterial strain, 26 strains of 4b type bacterial strain, 10 strains of 4a type bacterial strain, 4c type bacterial strain 2 strains (referring to table 1).
1.3PCR Establishing
1.3.1 primer screening
Adopt comparative genomics method, and with reference to pertinent literature, conserved regions and variable region in the special gene region of serotype are compared, design 8 primer L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F, L4-R.Primer sequence refers to table 2.
1.3.2PCR reaction system and condition
By the optimization of PCR system and condition, determine multi-PRC reaction system (ddH
2o is diluent) be: 10 * Taq Buffer is (containing Mg
2+) 3 μ L, 10mmol/L dNTP Mix0.6 μ L, each 0.5 μ L of 50 μ M primers, Taq DNA polymerase0.4 μ L, positive control solution 5 μ L or sample to be checked 5 μ L, lysate 10 μ L, add ddH
2o supplies volume to 30 μ L; Reaction conditions is: 95 ℃ of 3min; 95 ℃ of 45s, 55 ℃ of 30s, 72 ℃ of 55s, 25 circulations; 72 ℃ of 5min.Product is electrophoresis in 1% sepharose 0.5 * tbe buffer liquid, after Goldview dyeing, with gel imaging system, analyzes.
1.3.3PCR product order-checking is identified
The rubber tapping of PCR product is reclaimed, be cloned into pMD18-T carrier, be transformed into DH5 α, after extraction plasmid, identify, positive colony is delivered to the order-checking of Shanghai Ying Jun Bioisystech Co., Ltd.Sequence, through NCBI BLAST contrast, be take and confirmed fragment as object band.
1.3.4 specific test
With listera innocua (Listeria innocua), listeria ivanovii (Listeria ivanovii), Wei Shi listeria bacteria (Listeria welshimeri), Sai Shi listeria bacteria (Listeria seeligeri), listeria grayi (Listeria grayi), subtilis (Bacillus subtilis), bacillus cereus (Bacillus cereus), enterococcus faecalis (Enterococcus faecalis), streptococcus aureus (Staphylococcus aureus), swine streptococcus (Streptococcus suis), streptococcus equi epizootic disease subspecies (Streptococcus.equi subsp.zooepidemicus), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus curvatus (Lactobacillus crispatus), Aeromonas hydrophila (Aeromonas hydrophila), Pseudomonas aeruginosa (Pseudomonas aeruginosa), vibrio alginolyticus (Vibrio alginolyticus), Vibrio parahaemolyticus (Vibrioparahaemolyticus), colon bacillus (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), Salmonella enteritidis (Salmonella enteritidis), white dysentery Salmonellas (Salmonella pullorum), shigella flexneri (Shigella flexneri), actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), pasteurella multocida (Pasteurella multocida), segmental bronchus sepsis rich for 25 kinds of bacteriums of Salmonella (Bordetella bronchiseptica) as negative control, distilled water is blank, by the PCR method of foundation, increases.ATCC is U.S.'s representative microbial preservation center; CMCC is Chinese medicine microbial strains preservation administrative center, and address is No. 2, Tiantan Xili, Beijing.
Table 1 listeria bacteria reference strain and negative control bacterial strain
Table 2 primer sequence and product length
1.3.5 susceptibility test
The representative strain of each serotype of Listeria monocytogenes (1/2a type bacterial strain EGD-e, 1/2b type bacterial strain 54004,1/2c type bacterial strain 54002,4b type strains A TCC19115) at BHI(Oxoid, Hampshire, England) in after 37 ℃ of incubated overnight, get 1mL bacterium liquid centrifugal and with PBS(0.01M, pH7.2) resuspended, adjust bacterial concentration to 10
8cFU/mL (OD
600be about 0.2), do 10 times of continuous gradient dilutions.Choose 10
-5, 10
-6, 10
-7, 10
-8extent of dilution carries out plate count, and gets each dilution bacterium liquid and carry out PCR detection.
The 1.4 food strain isolated somatotype of being correlated with
The relevant strain isolated separation of 300 strain Listeria monocytogenes food is from fishery products, milk preparation, meat product, vegetables and food processing plant's utensil in East & South China area, sewage etc.These strain isolateds all carry out somatotype with this test kit, and use serotype test kit based on agglutination reaction of serum (Japanese デ ンカSheng Yan Co., Ltd.) to identify simultaneously.
1.5 application of samples and detecting step
1.5.1 1.5mL diluent in cryopreservation tube G is added to cryopreservation tube A, resuspended, mix and make suspension, deposit in-20 ℃ standby;
1.5.2 the 10 μ L of the lysate in cryopreservation tube B are added respectively to PCR reaction tubes, be divided into contrast PCR reaction tubes and PCR reaction tubes to be checked, again the positive control solution 5 μ L in cryopreservation tube C-cryopreservation tube F are added respectively in contrast PCR reaction tubes, sample 5 μ L to be checked are added to PCR reaction tubes to be measured, mix, the suspension 15 μ L of cryopreservation tube A in step (1.5.1) are added to above-mentioned each PCR reaction tubes, carry out immediately pcr amplification;
1.5.3 PCR reaction tubes is increased on PCR instrument by following reaction conditions: 95 ℃ of 3min; 95 ℃ of 45s, 55 ℃ of 30s, 72 ℃ of 55s, 25 circulations; 72 ℃ of 5min;
1.5.4 by the product electrophoresis in 1% sepharose 0.5 * tbe buffer liquid after amplification, after Goldview dyeing with gel imaging system read, analytical results.
2 results
2.1 multiplex PCRs and test kit detected result
Utilize this test kit to increase to the main serotype of Listeria monocytogenes, serotype 1/2a can obtain the band (A in Fig. 1) of 691bp; 1/2b type can obtain the band (B in Fig. 1) of 471bp; 1/2c type can obtain 691bp and two bands of 906bp (C in Fig. 1) simultaneously; 4b type can obtain 471bp and two bands of 597bp (D in Fig. 1) simultaneously; Other serotypes outside 4 kinds of main serotypes (as 4a type and 4c type) all do not increase and obtain any band.
The order-checking of 2.2PCR product is identified
After pcr amplification product is compared with BLAST through checking order, result demonstration, each amplified band sequence is all 99%～100% with the homology of goal gene regional sequence, shows that this PCR method has higher accuracy.
2.3 specific test
Utilize this test kit respectively 25 kinds of other bacteriums to be increased, result shows without any band, shows to adopt the main serotype specificity fragment of the method amplification Listeria monocytogenes to have good specificity.
2.4 susceptibility tests
Utilize this test kit to detect each dilution listeria bacteria bacterium liquid, when each serotype is respectively containing having an appointment 10
2during the above bacterium of the CFU/mL order of magnitude, all can increase and obtain corresponding object band, show that the susceptibility of each main serotype of the method amplification Listeria monocytogenes all can reach 10
2cFU/mL.
The relevant strain isolated of 2.5 food is identified and species confirmation
In the relevant strain isolated of food in 300 strains from East & South China area, by the evaluation of this test kit, 143 strains are 1/2a type, and 78 strains are 1/2b type, and 51 strains are 1/2c type, and 28 strains are 4b type; And the coincidence rate of agglutination reaction of serum and this test kit somatotype result is 90.7%(272/300), difference appears at: the bacterial strain that 3 strain agglutination reaction are judged to be 1/2a type is 1/2b type in identification with multi-plex PCR; The bacterial strain that 2 strain agglutination reaction are judged to be 4c type is 4b type in identification with multi-plex PCR; Other 23 strains in agglutination reaction, occur cross reaction, cannot result of determination bacterial strain, identification with multi-plex PCR wherein 13 strains is 1/2a type, 4 strains are 1/2b type, 6 strains are 4b type.By improving thalline pre-treating process and re-starting agglutination reaction of serum, result is all supported multiplex PCR result.Above result confirms that the fast typing test kit based on multiplex PCR has higher stability.
Claims (6)
1. the main serotype fast typing of a Listeria monocytogenes test kit, it is characterized in that described test kit comprises cryopreservation tube and PCR reaction tubes, described cryopreservation tube comprises cryopreservation tube A, cryopreservation tube B, cryopreservation tube C, cryopreservation tube D, cryopreservation tube E, cryopreservation tube F and cryopreservation tube G, described cryopreservation tube A is equipped with the Taq archaeal dna polymerase for PCR reaction, 10 * buffer, lyophilized powder prepared by the mixed solution of dNTP mixture and primer, cryopreservation tube B is equipped with lysate, cryopreservation tube C, cryopreservation tube D, cryopreservation tube E, cryopreservation tube F is equipped with respectively Listeria monocytogenes 1/2a type positive control damping fluid, Listeria monocytogenes 1/2b type positive control damping fluid, Listeria monocytogenes 1/2c type positive control damping fluid, Listeria monocytogenes 4b type positive control damping fluid, cryopreservation tube G is equipped with diluent, described diluent is distilled water, in described cryopreservation tube A, the sequence of primer is:
L1-F：AGCAAAATGCCAAAACTCGT
L1-R：CATCACTAAAGCCTCCCATTG
L2-F：AGTGGACAATTGATTGGTGAA
L2-R：CATCCATCCCTTACTTTGGAC
L3-F：AGGGCTTCAAGGACTTACCC
L3-R：ACGATTTCTGCTTGCCATTC
L4-F：AGGGGTCTTAAATCCTGGAA
And L4-R:CGGCTTGTTCGGCATACTTA.
2. the main serotype fast typing of Listeria monocytogenes test kit as claimed in claim 1, is characterized in that described PCR reaction tubes is 100～300.
3. the main serotype fast typing of Listeria monocytogenes test kit as claimed in claim 1, it is characterized in that the lyophilized powder in described cryopreservation tube A is first at-80 ℃ of pre-freeze 10h by described mixed solution in cryopreservation tube A, again at-40 ℃ of lasting freeze-drying 30h, then with the speed of 1.5 ℃/min, be warming up to 25 ℃ of dry 2h moisture content is reached below 5%, obtain lyophilized powder; Described every 8 μ L mixed solutions consist of: Taq archaeal dna polymerase 0.4 μ L, 10 * buffer3 μ L, dNTP mixture0.6 μ L, concentration is each 0.5 μ L of L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R primer of 50 μ M, and dATP, dCTP, dGTP and dTTP that described dNTP mixture is 10mM by final concentration form.
4. the main serotype fast typing of Listeria monocytogenes test kit as claimed in claim 1, is characterized in that described in cryopreservation tube B, lysate final concentration consists of: volumetric concentration 4%Triton-X100,5mg/mLNaN
3, 1mol/L Tris, pH is 8.0, solvent is distilled water.
5. the main serotype fast typing of Listeria monocytogenes test kit as claimed in claim 1, it is characterized in that each test kit has 100 PCR reaction tubess, in described cryopreservation tube A the amount of lyophilized powder with freeze-drying before the volume of mixed solution count 800 μ L, in described cryopreservation tube B, lysate 1mL is housed, cryopreservation tube C, cryopreservation tube D, cryopreservation tube E, cryopreservation tube F are equipped with positive control damping fluid 0.5mL separately, and cryopreservation tube G is equipped with 1.5mL diluent.
6. a method of utilizing the main serotype fast typing of Listeria monocytogenes test kit described in claim 1 to carry out the main Serotypes of Listeria monocytogenes, it is characterized in that described classifying method carries out as follows: (1) adds to cryopreservation tube A by diluent in cryopreservation tube G, resuspended, mix and make suspension, deposit in-20 ℃ standby; Described lyophilized powder consumption in freeze-drying before mixeding liquid volume, in described cryopreservation tube G, diluent and mixeding liquid volume are than being 15:8; (2) lysate in cryopreservation tube B is added respectively to PCR reaction tubes, be divided into contrast PCR reaction tubes and PCR reaction tubes to be checked, successively the positive control damping fluid in cryopreservation tube C, cryopreservation tube D, cryopreservation tube E and cryopreservation tube F is added in corresponding contrast PCR reaction tubes again, sample to be checked is added to PCR reaction tubes to be checked, mix respectively, and then add the suspension in step (1) cryopreservation tube A in each PCR reaction tubes, carry out immediately pcr amplification; Described sample to be checked is that Listeria monocytogenes to be checked is seeded in BHI substratum, after 37 ℃ of overnight incubation, gets 100 ℃ of bacterium liquid and boils centrifugal abandoning supernatant after deactivation, gets precipitation resuspended with distilled water, makes sample to be checked; In described cryopreservation tube B, in lysate and cryopreservation tube A, the volume ratio of suspension is 2:3, the volume ratio of the positive control damping fluid in described cryopreservation tube B in lysate and cryopreservation tube C, cryopreservation tube D, cryopreservation tube E and cryopreservation tube F is 2:1, and in described cryopreservation tube B, lysate is 2:1 with sample volume ratio to be checked;
(3) each PCR reaction tubes of step (2) is increased on PCR instrument by following reaction conditions: 95 ℃ of preheating 3min; 95 ℃ of sex change 45s, 55 ℃ of annealing 30s, 72 ℃ are extended 55s, 25 circulations; 72 ℃ keep 5min;
(4) by the electrophoresis in mass concentration 1% sepharose and 0.5 * tbe buffer liquid respectively of the product after step (3) amplification, use gel imaging system reading of data record after Goldview dyeing;
(5) result is judged: after detection, what the positive control solution in sample to be checked and cryopreservation tube C amplified 691bp band simultaneously is the Listeria monocytogenes 1/2a type positive; What in sample to be checked and cryopreservation tube D, positive control solution amplified 471bp band simultaneously is the Listeria monocytogenes 1/2b type positive; What in sample to be checked and cryopreservation tube E, positive control solution amplified 691bp, two bands of 906bp simultaneously is the Listeria monocytogenes 1/2c type positive; What in sample to be checked and cryopreservation tube F, positive control solution amplified 471bp, two bands of 597bp simultaneously is the Listeria monocytogenes 4b type positive; Positive control solution there is band and sample to be checked without band for the main serotype of non-Listeria monocytogenes; All without band or sample to be checked, there is band and positive control solution need again detect and judge without band in sample to be checked and positive control solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310571311.9A CN103602739A (en) | 2013-11-13 | 2013-11-13 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310571311.9A CN103602739A (en) | 2013-11-13 | 2013-11-13 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103602739A true CN103602739A (en) | 2014-02-26 |
Family
ID=50121002
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310571311.9A Pending CN103602739A (en) | 2013-11-13 | 2013-11-13 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103602739A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218805A (en) * | 2019-06-11 | 2019-09-10 | 扬州大学 | Listeria monocytogenes serotype 4h multiple PCR detection kit |
| CN110423833A (en) * | 2019-08-28 | 2019-11-08 | 华南理工大学 | A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype |
| 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 |
| CN112763640A (en) * | 2019-11-06 | 2021-05-07 | 中国检验检疫科学研究院 | Novel method for identifying four serotype differential metabolites of listeria monocytogenes |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286612A (en) * | 2010-06-18 | 2011-12-21 | 中国科学院上海生命科学研究院 | Fast detection reagent kit for pathogenic microorganism |
| CN102321762A (en) * | 2011-09-14 | 2012-01-18 | 上海德诺产品检测有限公司 | Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof |
| CN102676647A (en) * | 2012-02-13 | 2012-09-19 | 东北农业大学 | Kit for detecting Listeria monocytogenes and staphylococcus aureus, and application thereof |
| CN102827931A (en) * | 2012-08-15 | 2012-12-19 | 广东出入境检验检疫局检验检疫技术中心 | Detection primer for detecting listeria monocytogenes by PCR-pyrophosphate method, kit and detection method |
| CN102851381A (en) * | 2012-09-21 | 2013-01-02 | 武汉真福医药科技发展有限公司 | LAMP kit for rapid detection of Listeria monocytogenes |
| CN103290119A (en) * | 2013-05-21 | 2013-09-11 | 南京师范大学 | Quintuple PCR (polymerase chain reaction) rapid detection method for main pathogenic bacteria in pork |
-
2013
- 2013-11-13 CN CN201310571311.9A patent/CN103602739A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286612A (en) * | 2010-06-18 | 2011-12-21 | 中国科学院上海生命科学研究院 | Fast detection reagent kit for pathogenic microorganism |
| CN102321762A (en) * | 2011-09-14 | 2012-01-18 | 上海德诺产品检测有限公司 | Method for rapid detection of listeria monocytogenes with high sensitivity and kit thereof |
| CN102676647A (en) * | 2012-02-13 | 2012-09-19 | 东北农业大学 | Kit for detecting Listeria monocytogenes and staphylococcus aureus, and application thereof |
| CN102827931A (en) * | 2012-08-15 | 2012-12-19 | 广东出入境检验检疫局检验检疫技术中心 | Detection primer for detecting listeria monocytogenes by PCR-pyrophosphate method, kit and detection method |
| CN102851381A (en) * | 2012-09-21 | 2013-01-02 | 武汉真福医药科技发展有限公司 | LAMP kit for rapid detection of Listeria monocytogenes |
| CN103290119A (en) * | 2013-05-21 | 2013-09-11 | 南京师范大学 | Quintuple PCR (polymerase chain reaction) rapid detection method for main pathogenic bacteria in pork |
Non-Patent Citations (1)
| Title |
|---|
| 郑华英等: "单增李斯特菌食品分离株多重PCR鉴定与分型", 《中国公共卫生》, vol. 25, no. 3, 31 March 2009 (2009-03-31), pages 332 - 333 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218805A (en) * | 2019-06-11 | 2019-09-10 | 扬州大学 | Listeria monocytogenes serotype 4h multiple PCR detection kit |
| CN110423833A (en) * | 2019-08-28 | 2019-11-08 | 华南理工大学 | A kind of multiple PCR method based on specific target identification Listeria monocytogenes serotype |
| CN112763640A (en) * | 2019-11-06 | 2021-05-07 | 中国检验检疫科学研究院 | Novel method for identifying four serotype differential metabolites of listeria monocytogenes |
| CN112763640B (en) * | 2019-11-06 | 2024-04-09 | 中国检验检疫科学研究院 | Novel method for identifying four serotypes of listeria monocytogenes differential metabolites |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhuang et al. | Detection of Salmonella spp. by a loop‐mediated isothermal amplification (LAMP) method targeting bcfD gene | |
| Geimba et al. | Serological characterization and prevalence of spvR genes in Salmonella isolated from foods involved in outbreaks in Brazil | |
| Zhang et al. | Development of a fimY-based loop-mediated isothermal amplification assay for detection of Salmonella in food | |
| Moosavy et al. | Detection of Salmonella spp in commercial eggs in Iran | |
| CN103602739A (en) | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit | |
| Karunasagar et al. | Listeria in tropical fish and fishery products | |
| Tao et al. | Investigation on prevalence of Listeria spp. and Listeria monocytogenes in animal-derived foods by multiplex PCR assay targeting novel genes | |
| Heo et al. | Rapid detection of Listeria monocytogenes by real-time PCR in processed meat and dairy products | |
| CN103667498A (en) | Detection method for vibrio parahemolyticus | |
| Barbuddhe et al. | The genus listeria | |
| Jeshveen et al. | Optimization of multiplex PCR conditions for rapid detection of Escherichia coli O157: H7 virulence genes. | |
| CN104726581A (en) | Vibrio parahaemolyticus constant-temperature detection primer set, detection kit and detection method capable of avoiding false negative detection result | |
| Xie et al. | Simultaneous enumeration of Cronobacter sakazakii and Staphylococcus aureus in powdered infant foods through duplex TaqMan real-time PCR | |
| Amin et al. | Molecular Characterization of Salmonella Enterica Isolated from Chicken Meat and its Products by Multiplex PCR. | |
| PURWANINGSIH et al. | Detection of Salmonella typhimurium ATCC 14028 in supplement health product liquid preparation using Real-Time PCR (qPCR) | |
| Catarame et al. | Comparison of a real‐time polymerase chain reaction assay with a culture method for the detection of Salmonella in retail meat samples | |
| CN108034740B (en) | Primer group for detecting Salmonella indiana, application of primer group, kit containing primer group and detection method of primer group | |
| Zhang et al. | Simple and rapid detection of Salmonella by direct PCR amplification of gene fimW | |
| Ohud et al. | Detection of Salmonella strains in clinical samples from Saudi Arabia by invA and hilA polymerase chain reaction (PCR)-based assays | |
| CN111020039B (en) | Campylobacter jejuni species specific molecular target and rapid detection method thereof | |
| WO2020248819A1 (en) | Multiplex pcr detection kit of listeria monocytogenes serotype 4h | |
| Wang et al. | Rapid, sensitive, specific, and visual detection of salmonella in retail meat with loop-mediated isothermal amplification, targeting the invA gene | |
| Adiguzel et al. | Molecular characterization of Escherichia coli O157: H7 from retail beef in Erzurum, Turkey | |
| Costa et al. | Enterobacteria identification and detection of diarrheagenic Escherichia coli in a Port Complex | |
| Shang et al. | Novel multiplex PCR assays for rapid identification of Salmonella serogroups B, C1, C2, D, E, S. enteritidis, and S. typhimurium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140226 |