CN113957162A - Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology - Google Patents
Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology Download PDFInfo
- Publication number
- CN113957162A CN113957162A CN202111020719.8A CN202111020719A CN113957162A CN 113957162 A CN113957162 A CN 113957162A CN 202111020719 A CN202111020719 A CN 202111020719A CN 113957162 A CN113957162 A CN 113957162A
- Authority
- CN
- China
- Prior art keywords
- primer
- raa
- probe
- salmonella
- fluorescent
- 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
- 241000607142 Salmonella Species 0.000 title claims abstract description 87
- 239000000523 sample Substances 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 63
- 238000005516 engineering process Methods 0.000 title claims abstract description 22
- 230000003321 amplification Effects 0.000 claims abstract description 53
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 53
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 235000013305 food Nutrition 0.000 claims description 10
- 239000008213 purified water Substances 0.000 claims description 10
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 9
- 239000011654 magnesium acetate Substances 0.000 claims description 9
- 229940069446 magnesium acetate Drugs 0.000 claims description 9
- 235000011285 magnesium acetate Nutrition 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 62
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 108020004414 DNA Proteins 0.000 description 27
- 235000015278 beef Nutrition 0.000 description 13
- 238000012136 culture method Methods 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 239000008223 sterile water Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 102000018120 Recombinases Human genes 0.000 description 6
- 108010091086 Recombinases Proteins 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 4
- 101710116602 DNA-Binding protein G5P Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 101710162453 Replication factor A Proteins 0.000 description 4
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 description 4
- 101710176276 SSB protein Proteins 0.000 description 4
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 4
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- DIRFUJHNVNOBMY-UHFFFAOYSA-N fenobucarb Chemical compound CCC(C)C1=CC=CC=C1OC(=O)NC DIRFUJHNVNOBMY-UHFFFAOYSA-N 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 241000972773 Aulopiformes Species 0.000 description 3
- 241001135265 Cronobacter sakazakii Species 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 241000194032 Enterococcus faecalis Species 0.000 description 3
- 241000588915 Klebsiella aerogenes Species 0.000 description 3
- 241000588747 Klebsiella pneumoniae Species 0.000 description 3
- 241000186779 Listeria monocytogenes Species 0.000 description 3
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 3
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 229940092559 enterobacter aerogenes Drugs 0.000 description 3
- 229940032049 enterococcus faecalis Drugs 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 235000019515 salmon Nutrition 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 206010004022 Bacterial food poisoning Diseases 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 206010016952 Food poisoning Diseases 0.000 description 2
- 208000019331 Foodborne disease Diseases 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001138501 Salmonella enterica Species 0.000 description 2
- 241000531795 Salmonella enterica subsp. enterica serovar Paratyphi A Species 0.000 description 2
- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 238000012197 amplification kit Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000013330 chicken meat Nutrition 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 235000019688 fish Nutrition 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 238000009629 microbiological culture Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical group CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 1
- 241000697872 Bactria Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000190687 Gobius Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241000135164 Timea Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 229960001506 brilliant green Drugs 0.000 description 1
- HXCILVUBKWANLN-UHFFFAOYSA-N brilliant green cation Chemical compound C1=CC(N(CC)CC)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](CC)CC)C=C1 HXCILVUBKWANLN-UHFFFAOYSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- C12Q1/68—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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of microbial detection, and particularly discloses a primer probe combination, a kit and a method for detecting salmonella based on a fluorescent RAA technology. The primer probe combination comprises a primer pair and a probe, wherein the primer pair comprises an upstream primer and a downstream primer, the sequence of the upstream primer is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.5, and the sequence of the probe is shown as SEQ ID NO. 3. The kit and the method are adopted for detecting the salmonella, amplification is carried out under the isothermal condition of 39 ℃, and a large temperature changing instrument is not needed; the method has the advantages of simple operation, good specificity, high sensitivity, high accuracy and low cost, and the reaction is finished within 20-30 min; the method can be used for laboratory detection and on-site rapid detection with simple resources, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a primer probe combination, a kit and a method for detecting salmonella based on a fluorescent RAA technology.
Background
Salmonella is a common food-borne pathogenic bacterium, and can cause people and animals to have food-borne intestinal disease symptoms such as diarrhea, vomiting and the like. According to statistics, in various bacterial food poisoning of various countries in the world, food poisoning caused by salmonella is often listed as the top, and food poisoning events caused by meat products and dairy products polluted by salmonella account for about 40% of the bacterial food poisoning events. Therefore, salmonella is a major concern in the public health field as an important index for food detection.
The traditional salmonella detection method is a microbial culture method, national standard GB 4789.4-2016 food safety national standard food microbiology inspection for salmonella inspection is formulated in China, and the detection of salmonella is standardized. However, the traditional detection method needs more than 5 days to obtain results, and the detection steps are complicated, time-consuming and labor-consuming. Therefore, in recent years, many researchers have conducted research and research on the rapid microbial detection technology.
Compared with a microbial culture method, the molecular biological method can effectively shorten the detection time, and the current molecular detection technology for salmonella mainly comprises PCR, real-time fluorescence PCR, LAMP, a gene chip and the like; however, these methods also have general disadvantages: the common PCR is complicated to operate and needs electrophoresis; the fluorescent PCR equipment is large and can only be used in a laboratory, and field detection cannot be carried out; LAMP isothermal amplification is fast in speed but low in specificity, and is easy to pollute; the detection sensitivity of the gene chip is often not high enough, and the cost is not very high.
Therefore, a sensitive, simple, rapid and low-cost salmonella detection method which can technically perform both laboratory detection and on-site rapid detection is still lacking.
Disclosure of Invention
In order to solve the problems, the invention provides a primer probe combination, a kit and a method for detecting salmonella based on a fluorescent RAA technology.
As a first aspect of the present invention, the present invention provides a primer probe combination for detecting salmonella based on a fluorescent RAA technique, comprising a primer pair and a probe, wherein the primer pair comprises an upstream primer and a downstream primer, and the sequence of the upstream primer is SEQ ID No. 1: CTGAAGTTGAGGATGTTATTCGCAAAGGGATCCG, the sequence of the downstream primer is SEQ ID NO. 5: TCGACGGACATCGACAGACGTAAGGAGGAC, the sequence of the probe is SEQ ID NO. 3: CTAATTTGATGGATCTCATTACACTTAAGT- (FAM-dT) -G-THF-A- (BHQ-dT) -GATTTATTGATTGCA. In the probe sequence, FAM-dT represents thymine nucleotide carrying a fluorescent group, THF represents tetrahydrofuran linker, BHQ-dT represents thymine nucleotide carrying a fluorescence quenching group, and C3 Spacer represents a Spacer.
The recombinase-mediated strand displacement nucleic acid amplification (RAA) is a new technology which can make trace nucleic acid be efficiently and rapidly amplified in vitro. The fluorescent RAA technique relies primarily on three enzymes: the recombinase UvsX, single-stranded DNA binding protein (SSB) and DNA polymerase. The recombinase UvsX is obtained from escherichia coli, the fluorescent RAA technology utilizes the recombinase UvsX to combine primers to anneal template DNA, SSB is combined to form a D ring structure, and the single-stranded state of the template DNA is kept under the action of DNA polymerase for amplification and extension. At the constant temperature of 37-42 ℃, the fluorescence signal can be obtained within 20 minutes, and the detection time is effectively shortened. And does not need temperature change in the nucleic acid amplification process, and is particularly suitable for operation on site and in places with simple resources. At present, recombinase isothermal amplification technology has been developed greatly and is applied to a plurality of detection fields of pathogen, food safety and the like.
As a second aspect of the invention, the invention provides a kit for detecting salmonella based on a fluorescent RAA technology, which comprises the primer probe combination.
Preferably, the kit for detecting salmonella based on the fluorescent RAA technology further comprises buffer, magnesium acetate and purified water. The buffer solution is a fluorescent RAA basic universal reagent, comprises components such as recombinase, single-stranded DNA binding protein, DNA polymerase, dNTP and the like, can be prepared by self, and can also be obtained by purchasing a commercial fluorescent RAA amplification reagent.
As a third aspect of the invention, the invention provides a method for detecting salmonella based on a fluorescence RAA technology, which comprises the following specific steps: extracting the genome DNA of a sample to be detected; performing fluorescent RAA amplification by using the kit for detecting salmonella based on the fluorescent RAA technology by using the genome DNA of a sample to be detected as a template; and analyzing whether the sample to be detected contains salmonella or not according to the fluorescent RAA amplification curve. Note: the fluorescent RAA amplification is real-time fluorescent RAA amplification.
Preferably, in the reaction system for fluorescent RAA amplification, the final concentrations of the upstream primer, the downstream primer and the probe are respectively 100-500 nmol/L. More preferably, the final concentration of the upstream primer, the downstream primer and the probe is 100-400 nmol/L. Most preferably, the final concentration of the upstream primer and the final concentration of the downstream primer are respectively 200nmol/L, and the final concentration of the probe is 400 nmol/L.
Preferably, the reaction system for the fluorescent RAA amplification is: 25 mu L of buffer solution, 1 mu L of each of the upstream primer and the downstream primer, 2 mu L of probe, 2.5 mu L of magnesium acetate, and 18.5 mu L of DNA template and purified water; the concentrations of the upstream primer, the downstream primer and the probe are respectively 10 mu M. Note: 10 μ M refers to the initial concentration of each primer and probe used.
Preferably, the reaction conditions for the fluorescent RAA amplification are: the temperature is 35-42 deg.C, and the time is 20-33 min. More preferably, the reaction conditions for the fluorescent RAA amplification are: the temperature is 39 ℃ and the time is 30 min.
As a fourth aspect of the invention, the invention also provides application of the method for detecting salmonella based on the fluorescence RAA technology in detecting salmonella in food, and the method can be used for detecting salmonella in food such as beef, chicken, salmon, and barcoo, and has a wide application range.
The invention has the following beneficial effects:
1. the specificity is good: when the kit and the method are used for detecting the salmonella, no cross reaction is generated with Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli, Staphylococcus aureus, enterococcus faecalis, hemolytic streptococcus and Enterobacter sakazakii.
2. The sensitivity is high: the sensitivity of detecting the salmonella is improved by pairing and screening of the primer and the probe and optimizing the concentration of the probe, and the detection limit of the salmonella in the actual sample detection by adopting the kit and the method is obviously lower than that of the traditional streak culture method.
3. The accuracy is high: the kit and the method provided by the invention are used for detecting salmonella in an actual sample, the detection result is basically consistent with that of the traditional streak culture method, and the reliability is high.
4. The operation is simple: the invention does not need large-scale temperature-changing equipment for detecting the salmonella, and does not need complicated steps such as the denaturation of double-stranded DNA and the like in advance; only a constant-temperature fluorometer is needed, the condition is mild, the reaction is simple, convenient and quick, and the method is not only suitable for laboratory detection, but also suitable for simple and easy resource field detection.
5. The cost is low: compared with the existing molecular biology detection method, the method has the advantages that the instrument, time and labor cost are obviously reduced, and the method has good application prospect.
Drawings
FIG. 1: the result of the salmonella fluorescence RAA amplification at 30 ℃ and 35 ℃;
FIG. 2: the result of the fluorescent RAA amplification of the salmonella at 39 ℃ and 42 ℃;
FIG. 3: salmonella fluorescence RAA amplification effect under different primer concentrations and different probe concentrations;
FIG. 4: the fluorescence RAA test results of the primer probe combination F3R3P3 and F3R4P 3;
FIG. 5: the fluorescence RAA test results of the primer probe combination F4R3P3 and F4R4P 3;
FIG. 6: the fluorescence RAA specificity test result of the primer probe combination F3R4P 3;
FIG. 7: the results of the fluorescent RAA sensitivity test for salmonella ATCC 14028;
FIG. 8: fluorescence RAA detection results of salmonella with different concentrations in beef after different enrichment times;
FIG. 9: and (4) carrying out fluorescence RAA detection results after different enrichment times of salmonella with different concentrations in the basha.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1: reaction parameter optimization and primer probe screening
First, primer Probe design
Obtaining Salmonella from NCBI GenBank databaseinvAAnd (3) gene sequence, and designing fluorescent RAA primer and probe sequence according to RAA primer and probe design principle. The primers and the probes are synthesized by Shanghai biological engineering Co., Ltd, and the specific sequences are shown in the following table 1:
TABLE 1 Salmonella fluorescent RAA primer Probe sequences
Second, reaction temperature optimization
Optimizing the reaction temperature by taking primer probes Sal-invA-F3, Sal-invA-R3 and Sal-invA-P3 as a combination, and performing fluorescent RAA amplification by adopting an RAA-1620 fluorescent RAA detector (Jiangsu Qitian gene biotechnology limited) at different temperatures (30 ℃, 35 ℃, 39 ℃ and 42 ℃) for 30 min; the reaction system (refer to fluorescence RAA amplification kit of Qitian gene biotechnology limited of Jiangsu) is as follows: 25 μ L of buffer, 2.1 μ L of each of the forward primer and the reverse primer (10 μ M), 0.6 μ L of probe (10 μ M), 2.5 μ L of magnesium acetate, 17.7 μ L of purified water for DNA template, and a final volume of 50 μ L; the DNA template is salmonella genomic DNA. As shown in FIGS. 1-2 (in the figure, well2 is an amplification well, and well3 is a blank control well), the amplification fluorescence value was the highest at 39 ℃ and the optimal amplification temperature was 39 ℃.
Third, optimization of primer probe concentration
Adjusting the addition amounts of the primer, the probe and the purified water in the basic reaction system for the fluorescent RAA amplification, and respectively diluting the final concentrations of the primer to 100nmol/L, 200nmol/L, 300nmol/L and 400nmol/L when the final concentration of the probe is 120 nmol/L; when the final concentrations of the primers are 200nmol/L and 400nmol/L, the corresponding final concentrations of the probes are respectively diluted to 100nmol/L, 200nmol/L, 300nmol/L and 400nmol/L for amplification; the reaction conditions are as follows: the temperature is 39 ℃ and the time is 30 min; and (4) screening the primer probe concentration under the optimal amplification effect. As shown in FIG. 3, the amplification signal was the best when the final concentration of the probe was 120nmol/L and the primer concentration was 200nmol/L (FIG. 3-1 shows the fluorescent RAA amplification structure with different primer concentrations when the final concentration of the probe was 120 nmol/L); while, in the case of the primer concentration being unchanged, the amplification signal also increases with the increase of the probe concentration (FIG. 3-2 shows the fluorescent RAA amplification structure at different probe concentrations at a primer concentration of 200nmol/L, and FIG. 3-3 shows the fluorescent RAA amplification structure at different probe concentrations at a primer concentration of 400 nmol/L). Therefore, the optimal final concentration of the primer is 200nmol/L and the optimal final concentration of the probe is 400nmol/L, and the reaction system corresponding to the final concentration is as follows: 25 μ L of buffer solution, 1 μ L of each of the forward primer (10 μ M) and the reverse primer (10 μ M), 2 μ L of probe (10 μ M), 2.5 μ L of magnesium acetate, and 18.5 μ L of DNA template and purified water as the optimized reaction system of the present invention.
Fourth, screening optimal primer probe combination
The primers Sal-invA-F3, Sal-invA-R3, Sal-invA-F4 and Sal-invA-R4 in Table 1 were combined to obtain a probe of Sal-invA-P3, and the amplification effects of the primer probe combinations of F3R3P3, F3R4P3, F4R3P3 and F4R4P3 were tested. The reaction system is as follows: 25 mu L of buffer solution, 1 mu L of each of an upstream primer (10 mu M) and a downstream primer (10 mu M), 2 mu L of probe (10 mu M), 2.5 mu L of magnesium acetate, 4 mu L of DNA template and 14.5 mu L of purified water, wherein the DNA template is salmonella genomic DNA with gradient concentration; the reaction conditions are as follows: the temperature is 39 ℃ and the time is 30 min. As a result, as shown in FIGS. 4 to 5, it can be seen that the fluorescence value amplified under the primer probe pair F3R4P3 was the highest, and the concentration of the added DNA template was 3X 102Since the fluorescence value at CFU/mL was still around 13500 mV, the detection limit was low, F3R4P3 was selected as the primer probe combination for amplification.
Example 2: detection kit and detection method for detecting salmonella based on fluorescence RAA technology
According to the results of primer probe screening of example 1, this example provides a kit for detecting salmonella based on the fluorescent RAA technique, the kit comprising: an upstream primer Sal-invA-F3, a downstream primer Sal-invA-R4, a probe Sal-invA-P3, a buffer solution, magnesium acetate and purified water, wherein the buffer solution is purchased from a fluorescent RAA amplification kit of Qitian gene biotechnology limited of Jiangsu.
According to the optimized result of the reaction parameters in the embodiment 1, the embodiment also provides a method for detecting salmonella based on the fluorescence RAA technology, which comprises the following specific steps:
a. extracting the genome DNA of a sample to be detected;
b. performing real-time fluorescence RAA amplification by using the kit for detecting salmonella based on the fluorescence RAA technology in the embodiment by using the genome DNA of a sample to be detected as a template; the reaction system of the fluorescent RAA amplification is as follows: 25 μ L of buffer, 1 μ L each of the forward primer (10 μ M) and the reverse primer (10 μ M), 2 μ L of probe (10 μ M), 2.5 μ L of magnesium acetate, and 18.5 μ L of DNA template and purified water; the reaction conditions are as follows: the temperature is 39 ℃ and the time is 30 min;
c. and analyzing whether the sample to be detected contains salmonella or not according to the real-time fluorescent RAA amplification curve.
The following effect evaluations were performed on the detection kit and the detection method for detecting salmonella based on the fluorescent RAA technique, which were provided in example 2:
first, specificity detection
Selecting salmonella typhimurium ATCC14028 and other 5 salmonella standard strains: salmonella paratyphi ATCC9150, salmonella choleraesuis ATCC7001, salmonella enteritidis ATCC17036, salmonella enteritidis ATCC49216, and salmonella typhi CMCC 50071; and 9 salmonella isolates S1, S2, S3, S4, S5, S6, S7, S9; in addition, Listeria monocytogenes ATCC7644, Pseudomonas aeruginosa ATCC27853, Klebsiella pneumoniae ATCC13883, Enterobacter aerogenes ATCC13048, Escherichia coli ATCC29522, Staphylococcus aureus ATCC6538, enterococcus faecalis ATCC49452, Streptococcus hemolyticus ATCC21059, Enterobacter sakazakii ATCC 29544; after a DNA template is extracted from the pure culture of the strain, fluorescence RAA detection is carried out to verify the specificity of the method.
All strains were stored in 10% (w/v) glycerol broth at-80 ℃ and the recovery and DNA extraction methods of the strains were as follows:
recovering the strain: taking 100 mu L of bacterial liquid from a glycerol cryopreservation tube, inoculating the bacterial liquid into 5mL of brain-heart infusion culture broth, and oscillating at 37 ℃ and 150 r/min overnight;
preparing a DNA template: 1mL of bacterial culture 1.5mL of EP centrifuge tube is taken, centrifuged at 12000r/min for 10min to collect bacterial liquid, and supernatant is discarded; extracting bacterial DNA according to a bacterial genome DNA extraction kit method; storing at-20 deg.C for use.
The fluorescence RAA amplification result is shown in FIG. 6, in FIG. 6-1, Salmonella typhimurium; 2. salmonella paratyphi; 3. salmonella choleraesuis; 4. salmonella typhi; 5. salmonella enteritidis (ATCC 17036); 6. salmonella enteritidis (ATCC 49216); 7. listeria monocytogenes; 8. pseudomonas aeruginosa; 9. klebsiella pneumoniae; 10. enterobacter aerogenes; 11. escherichia coli; 12. staphylococcus aureus bacteria; 13. enterococcus faecalis; 14. hemolytic streptococcus; 15. enterobacter sakazakii; 16. sterile water.
In fig. 6-2, 1, S1; 2. s2; 3. s3; 4. s4; 5. s5; 6. s6; 7. s7; 8. s8; 9. sterile water.
As can be seen, only Salmonella typhimurium ATCC14028, the rest 5 Salmonella standard strains (figure 6-1) and the 9 Salmonella experimental isolates S1-S9 (figure 6-2) amplify fluorescence signals, and the rest 9 bacteria and sterile water which are not Salmonella do not amplify signals (figure 6-1), so that the primer probe combination F3R4P3 has good specificity, can amplify fluorescence signals for all the tested Salmonella and can be suitable for detecting the Salmonella.
Second, sensitivity detection
A single colony of the newly grown salmonella ATCC14028 on a nutrient agar plate is picked and inoculated in LB broth, and after 24 hours, 1mL of bacterial suspension is sequentially absorbed in 9 mL of LB broth to prepare a 10-fold gradient dilution. 1mL of the bacterial suspension was taken from each dilution tube to extract DNA, and the fluorescent RAA reaction was performed (the temperature after optimization of the fluorescent RAA reaction conditions, i.e., 39 ℃ C., final concentrations of the primers and probes were 200nmol/L and 400nmol/L, respectively), so that the lowest dilution at which a normal curve could be amplified was observed. And respectively coating 100 mu L of bacterial liquid on a flat plate, culturing for 24-48 h at 37 +/-1 ℃, counting bacterial liquids with different gradients, and calculating the concentration of the salmonella culture suspension so as to calculate the detection sensitivity of the fluorescence RAA method.
The fluorescence RAA amplification result is shown in FIG. 7-1, (FIG. 7-1, the fluorescence RAA sensitivity test result of Salmonella when the probe concentration is 400 nmol/L) shows that the fluorescence RAA amplification of Salmonella by the primer probe combination F3R4P3 can reach 3 × 101 CFU/mL. In addition, it was found through experiments that the influence of the probe concentration on the reaction sensitivity was very large, as shown in FIG. 7-2 (FIG. 7-2, the result of the Salmonella fluorescence RAA sensitivity test at a probe concentration of 120 nmol/L), and when the final probe concentration was 120nmol/L of the basic reaction, the sensitivity of the fluorescence RAA was only 3X 103 CFU/mL。
Third, testing the detection limit of the substrate
In order to verify the detection limit of the method on the salmonella in the actual sample, beef and the basha are selected from Shanghai markets and detected according to a traditional method and a fluorescence RAA amplification method respectively, wherein the traditional method is a streak culture method specified in GB 4789.4-2016 (national food safety Standard for food microbiology inspection) for detecting the salmonella.
The specific operation is as follows: determination of initial bacterial liquid concentration to 3X 10 by turbidimetry8CFU/mL, run 10-1To 10-10And (6) diluting. Weighing 25g of sample, adding salmonella pure bacteria liquid with different dilutions, adding the artificially polluted sample into 225 mL of BPW pre-enrichment medium, culturing at 36 ℃ for 18h, and adding 1mL of pre-enrichment liquid into 10 mL of sodium tetrasulfosulfonate brilliant green (TTB) enrichment liquid. Culturing at 42 deg.C for 2 and 24 hr, collecting 1mL culture solution, extracting DNA template, amplifying by fluorescence RAA method, and observing the result. And simultaneously, carrying out parallel detection on the bacterium liquid with the bacterium increment of 2h and 24h by using a traditional streak culture method.
The detection limit test results are as follows:
(1) comparison of detection limits of Salmonella in beef by fluorescence RAA method and traditional method
In beef sampleAs shown in FIG. 8 and Table 2, the results of the fluorescent RAA detection after different enrichment times and the results of the conventional streaking culture in artificial contamination with Salmonella of different concentrations are shown, and it can be seen that the limit of the fluorescent RAA amplification detection before BPW enrichment is 3X 103 CFU/mL (FIG. 8-1, fluorescent RAA amplification result before beef BPW enrichment; FIG. 8-1, 3X 107 CFU/mL；2、3×106 CFU/mL；3、3×105 CFU/mL；4、3×104 CFU/mL；5、3×103 CFU/mL；6、3×102 CFU/mL；7、3×101 CFU/mL; 8. sterile water. ) However, after 2h of enrichment for the 2 nd time of TTB, the detection limit of the fluorescent RAA on the salmonella in the beef can reach the original addition concentration of 3 multiplied by 10-2 CFU/mL (FIG. 8-2, fluorescent RAA amplification result after 2h of beef TTB enrichment), at which time the detection limit of the traditional streak culture method is 3 CFU/mL (Table 2); after TTB enrichment for 24h, the detection limit of salmonella in beef by fluorescent RAA is 3 multiplied by 10 of the original addition concentration-2 CFU/mL, the amplification value is higher than TTB enrichment for 2h (FIG. 8-3, fluorescent RAA amplification result after beef TTB enrichment for 24 h; in FIG. 8-3, 1, 3 × 106 CFU/mL；2、3×105 CFU/mL；3、3×104 CFU/mL；4、3×103 CFU/mL；5、3×102 CFU/mL；6、3×101 CFU/mL；7、3×100 CFU/mL；8、3×10-1 CFU/mL；9、3×10-2 CFU/mL; 10. sterile water) when the detection limit of the traditional streak culture method reaches 3X 10-1 CFU/mL (Table 2). Therefore, the sensitivity and the detection speed of the fluorescence RAA method for detecting the salmonella in the beef are obviously higher than those of the traditional streak culture method.
TABLE 2 comparison of the results of the fluorescence RAA method and the conventional method after adding Salmonella in beef for different enrichment times
(2) Comparison of detection limits of Salmonella in Bass by fluorescence RAA method and conventional method
Salmonella artificially polluted in different concentrations in the Bass fish sample, and fluorescence RAA detection junction after different bacteria increasing timeAs shown in FIG. 9 and Table 3, the results of the conventional streaking culture showed that the detection limit of the fluorescent RAA amplification before the enrichment of BPW was 3X 103 CFU/mL (FIG. 9-1, fluorescent RAA amplification result before BPW enrichment of Bashan salmon; FIG. 9-1, 3X 107 CFU/mL；2、3×106 CFU/mL；3、3×105 CFU/mL；4、3×104 CFU/mL；5、3×103 CFU/mL；6、3×102 CFU/mL；7、3×101 CFU/mL; 8. sterile water); after TTB enrichment only needs 2h, the detection limit of the fluorescent RAA on the salmonella in the Bassa can reach the original addition concentration of 3 multiplied by 10-2CFU/mL (FIG. 9-2, fluorescent RAA amplification result after 2h of Bactria gobius TTB enrichment), at which the detection limit of the traditional streaking culture method is 3 CFU/mL (Table 3); after TTB enrichment for 24h, the detection limit of the fluorescent RAA on the salmonella in the Bassa is 3 multiplied by 10 of the original addition concentration-2 CFU/mL, the amplification value was higher than TTB enrichment for 2h (FIG. 9-3, fluorescent RAA amplification result after 24h of TTB enrichment of Bassa fish; FIG. 9-3, 1, 3X 106 CFU/mL；2、3×105 CFU/mL；3、3×104 CFU/mL；4、3×103 CFU/mL；5、3×102 CFU/mL；6、3×101 CFU/mL；7、3×100 CFU/mL；8、3×10-1 CFU/mL；9、3×10-2 CFU/mL; 10. sterile water) when the detection limit of the traditional streak culture method reaches 3X 10-1 CFU/mL (Table 3). Therefore, the sensitivity and the detection speed of the fluorescence RAA method for detecting the salmonella in the basha are obviously higher than those of the traditional streak culture method.
TABLE 3 comparison of results of fluorescence RAA method and conventional method after adding Salmonella to Bassa and TTB enrichment
Fourthly, detecting actual samples
50 parts of beef, chicken, salmon and barbeque sold in Shanghai are pretreated and enriched according to GB 4789.4-2016, genome DNA is extracted, the established fluorescent RAA method is adopted for detection, and meanwhile, the traditional streak culture method recommended in GB 4789.4-2016 is adopted for detection, and the results are shown in Table 4, so that the detection result of the fluorescent RAA method is consistent with the detection result of the existing national standard GB 4789.4-2016, and the accuracy is high.
TABLE 4 results of Salmonella detection in actual samples
In conclusion, the kit and the method for detecting the salmonella have good specificity and high accuracy, compared with the traditional streaking culture method, the kit and the method have better sensitivity, greatly shorten the detection time (the kit can obtain a result only by reacting for about 30min after enrichment, and can obtain a result only by culturing for 24h after enrichment by the traditional method), and have remarkable advantages.
This detailed description is to be construed as illustrative only and is not to be taken as limiting the invention, as any changes that may be made by a person skilled in the art after reading the present specification will be protected by the patent laws within the scope of the appended claims.
Sequence listing
<110> Shanghai customs animal and plant and food inspection and quarantine technology center
<120> primer probe combination, kit and method for detecting salmonella based on fluorescent RAA technology
<141> 2021-09-01
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 1
ctgaagttga ggatgttatt cgcaaaggga tccg 34
<210> 2
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 2
tcattttctt aataaatcga cggacatcga c 31
<210> 3
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is THF (tetrahydrofuran)
<400> 3
ctaatttgat ggatctcatt acacttaagt tgnatgattt attgattgca 50
<210> 4
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 4
tcattttctt aataaatcga cggacatcga c 31
<210> 5
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 5
tcgacggaca tcgacagacg taaggaggac 30
Claims (10)
1. A primer probe combination for detecting salmonella based on a fluorescent RAA technology is characterized in that: the primer pair comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is SEQ ID NO. 1: CTGAAGTTGAGGATGTTATTCGCAAAGGGATCCG, the sequence of the downstream primer is SEQ ID NO. 5: TCGACGGACATCGACAGACGTAAGGAGGAC, the sequence of the probe is SEQ ID NO. 3: CTAATTTGATGGATCTCATTACACTTAAGT- (FAM-dT) -G-THF-A- (BHQ-dT) -GATTTATTGATTGCA.
2. A kit for detecting salmonella based on a fluorescent RAA technology is characterized in that: comprising the primer probe combination of claim 1.
3. The kit for detecting salmonella based on the fluorescent RAA technique according to claim 2, wherein: buffer, magnesium acetate and purified water are also included.
4. A method for detecting salmonella based on a fluorescence RAA technology is characterized in that: the method comprises the following specific steps: extracting the genome DNA of a sample to be detected; performing fluorescent RAA amplification by using the kit of claim 3 and using the genomic DNA of a sample to be detected as a template; and analyzing whether the sample to be detected contains salmonella or not according to the fluorescent RAA amplification curve.
5. The method for detecting salmonella based on the fluorescent RAA technique of claim 4, wherein: in the reaction system of the fluorescent RAA amplification, the final concentrations of the upstream primer, the downstream primer and the probe are respectively 100-400 nmol/L.
6. The method for detecting salmonella based on the fluorescent RAA technique of claim 5, wherein: the final concentrations of the upstream primer and the downstream primer are respectively 200 nmol/L; the final concentration of the probe was 400 nmol/L.
7. The method for detecting salmonella based on the fluorescent RAA technique of claim 5, wherein: the reaction system of the fluorescent RAA amplification is as follows: 25 mu L of buffer solution, 1 mu L of each of the upstream primer and the downstream primer, 2 mu L of probe, 2.5 mu L of magnesium acetate, and 18.5 mu L of DNA template and purified water; the concentrations of the upstream primer, the downstream primer and the probe are respectively 10 mu M.
8. The method for detecting salmonella based on the fluorescent RAA technique of claim 4, wherein: the reaction conditions for the fluorescent RAA amplification are as follows: the temperature is 35-42 deg.C, and the time is 20-33 min.
9. The method for detecting salmonella based on the fluorescent RAA technique of claim 8, wherein: the reaction conditions for the fluorescent RAA amplification are as follows: the temperature is 39 ℃ and the time is 30 min.
10. Use of the method for detecting salmonella based on the fluorescent RAA technique according to claims 4-9 for detecting salmonella in food products.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111020719.8A CN113957162A (en) | 2021-09-01 | 2021-09-01 | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111020719.8A CN113957162A (en) | 2021-09-01 | 2021-09-01 | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113957162A true CN113957162A (en) | 2022-01-21 |
Family
ID=79460766
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111020719.8A Pending CN113957162A (en) | 2021-09-01 | 2021-09-01 | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113957162A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634580A (en) * | 2012-04-05 | 2012-08-15 | 华南农业大学 | Primers and method for simultaneously detecting various pathogenic bacteria |
| CN108588251A (en) * | 2018-06-29 | 2018-09-28 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | RPA primers, probe and detection method for detecting salmonella |
| CN112553355A (en) * | 2020-12-29 | 2021-03-26 | 上海海关动植物与食品检验检疫技术中心 | Primer probe combination, kit and method for detecting listeria monocytogenes based on RAA technology |
| CN112760395A (en) * | 2021-03-01 | 2021-05-07 | 济南国益生物科技有限公司 | Primer probe set, kit and detection method for multiple detection of intestinal pathogenic bacteria based on fluorescence RMA method |
-
2021
- 2021-09-01 CN CN202111020719.8A patent/CN113957162A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634580A (en) * | 2012-04-05 | 2012-08-15 | 华南农业大学 | Primers and method for simultaneously detecting various pathogenic bacteria |
| CN108588251A (en) * | 2018-06-29 | 2018-09-28 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | RPA primers, probe and detection method for detecting salmonella |
| CN112553355A (en) * | 2020-12-29 | 2021-03-26 | 上海海关动植物与食品检验检疫技术中心 | Primer probe combination, kit and method for detecting listeria monocytogenes based on RAA technology |
| CN112760395A (en) * | 2021-03-01 | 2021-05-07 | 济南国益生物科技有限公司 | Primer probe set, kit and detection method for multiple detection of intestinal pathogenic bacteria based on fluorescence RMA method |
Non-Patent Citations (3)
| Title |
|---|
| LIWEI ZHAO等: "Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples", 《FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY》 * |
| 周冬根等: "沙门菌荧光重组酶介导等温扩增检测方法的应用研究", 《中国热带医学》 * |
| ***等: "重组酶介导扩增技术快速检测沙门菌方法的建立", 《中国国境卫生检疫杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kawasaki et al. | Multiplex real-time polymerase chain reaction assay for simultaneous detection and quantification of Salmonella species, Listeria monocytogenes, and Escherichia coli O157: H7 in ground pork samples | |
| CN103966353B (en) | A kind of detect the rugged Cronobacter sakazakii of slope method and test kit and primer | |
| CN113249499B (en) | Salmonella typhi detection kit, and preparation method and application thereof | |
| Chen et al. | Development of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Pseudomonas syringae pv. tomato in planta | |
| Yu et al. | A multipathogen selective enrichment broth for simultaneous growth of Salmonella enterica serovar Enteritidis, Staphylococcus aureus, and Listeria monocytogenes | |
| CN105936935B (en) | PCR detection kit for rapidly identifying specific serotype salmonella | |
| CN115747353A (en) | Primer group, reagent, kit and detection method for detecting Listeria monocytogenes | |
| Li et al. | Competitive annealing mediated isothermal amplification (CAMP) for rapid and simple detection of Listeria monocytogenes in milk | |
| Liu et al. | Sensitive dual readout assays based on rolling circle amplification for fluorescent and colorimetric detection of Cronobacter spp. in powdered infant formula | |
| Dong et al. | Loop-mediated isothermal amplification assay for sensitive and rapid detection of Phytophthora capsici | |
| Milton et al. | Novel saltatory rolling circle amplification assay for rapid and visual detection of Campylobacter jejuni in chicken meat | |
| Guo et al. | Multiple fluorescent saltatory rolling circle amplification (SRCA) for simultaneous and sensitive detection of Salmonella spp. and Shigella spp. in food | |
| CN104531860B (en) | A kind of molecular detecting method of Shigella and its application | |
| CN110699470A (en) | Dual PCR primer, kit, application and method for detecting salmonella and identifying pullorum disease/gallinarum serotype | |
| CN108179207B (en) | PCR primer and method for identifying different subgroup cucurbita bacterial fruit blotch germs | |
| CN113957162A (en) | Primer probe combination, kit and method for detecting salmonella based on fluorescence RAA technology | |
| CN108004334B (en) | Quadruple fluorescent PCR primer group, probe group, kit and method for detecting four pathogenic bacteria in drinking water | |
| CN113957164B (en) | CRISPR One post detection method and kit thereof for Cronobacter in infant formula powder | |
| CN112795674A (en) | Primer probe combination, kit and method for detecting Cronobacter sakazakii based on RAA technology | |
| CN115747351A (en) | Kit for detecting Shigella based on CRISPR/Cas12a method and use method thereof | |
| CN116121408A (en) | Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application | |
| CN104561343B (en) | Distinguish the primer and kit of detection low pathogenicity and highly pathogenic babesia motasi | |
| CN112553355A (en) | Primer probe combination, kit and method for detecting listeria monocytogenes based on RAA technology | |
| CN110804674B (en) | Primer probe composition and kit for detecting soybean root rot based on recombinase polymerase amplification method and application of primer probe composition and kit | |
| Milton et al. | Development and evaluation of a novel polymerase spiral reaction based testing technique for same-day visual detection of Campylobacter coli in pork |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220121 |