CN110982914A - Specific forward and reverse primers and probe for salmonella, detection kit and application of specific forward and reverse primers and probe - Google Patents
Specific forward and reverse primers and probe for salmonella, detection kit and application of specific forward and reverse primers and probe Download PDFInfo
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- CN110982914A CN110982914A CN201911311125.5A CN201911311125A CN110982914A CN 110982914 A CN110982914 A CN 110982914A CN 201911311125 A CN201911311125 A CN 201911311125A CN 110982914 A CN110982914 A CN 110982914A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention relates to the technical field of detection, and particularly relates to a salmonella specific forward and reverse primer, a probe, a detection kit and application thereof. The detection kit comprises a forward primer, a reverse primer, a probe and a lateral flow chromatography test strip; the 5' end of the reverse primer is marked by biotin; the 5 'end of the probe was labeled with Fluoroescein Isothiocyanate (FITC), the middle region C bases (at least 30bp from the 5' end and at least 15bp from the 3 'end) were replaced with [ THF ], and the 3' end was end-blocked with C3-spacer. The invention can realize real-time, real-time and instant rapid field detection in a non-laboratory environment independent of instruments and equipment by using an RPA-LFS (the isothermal amplification and the comparative flow strip) method, and has high accuracy.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a salmonella specific forward and reverse primer, a probe, a detection kit and application thereof.
Background
Salmonella is a gram-negative pathogenic bacterium widely distributed in nature, and food poisoning caused by salmonella is frequently listed among bacterial food poisoning of various countries in the world according to statistics. Salmonella is the first place in inland areas of China. The transmission pathway of salmonella is mainly the intake of food, such as contaminated meat, eggs and egg products, dairy products, seafood and the like. Salmonella is a great hazard to human and animal health, and human beings have symptoms of nausea, vomiting, diarrhea, abdominal pain or spasm, fever and headache after eating the contaminated food for 12-36 h. Therefore, the salmonella can be accurately, quickly and simply identified, so that the quality of food can be improved, the infection of the salmonella can be avoided, and the life safety of human can be ensured;
at present, the detection method of salmonella mainly comprises a traditional standard method, an immunological method, a PCR method, LAMP, a rolling circle amplification method and the like. The traditional standard method is used for increasing the bacteria of food samples step by step, aims to improve the detection rate of pathogenic bacteria, requires at least 4-7 days for detection, and is complex in detection process, time-consuming and labor-consuming. The immunological method is detection by utilizing the binding property of salmonella surface antigen and specific antibody. However, the method is easy to generate false positive, and the detection rate is only 103-. PCR has been widely used in the detection of Salmonella, and the qPCR primer designed for the stn enterotoxin gene of Salmonella by the Shinmiki et al, to obtain a 119bp fragment. Experiments show that the PCR has higher sensitivity to salmonella, and can detect 100 copies of standard plasmids. However, the method needs expensive instruments and equipment, has high requirements on the operation level of workers, and cannot meet the field detection requirements of basic medical organizations, remote areas and the like, so that the method for rapidly identifying the salmonella through the RPA-LFS technology is expected to bring new technical reference for the detection of the salmonella.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a salmonella specific forward and reverse primer, a probe, a detection kit and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a specific forward and reverse primer and a probe for salmonella, wherein the forward primer and the reverse primer are Inv-F, Inv-R respectively, and the sequences are
5'-CTACAAGCATGAAATGGCAGAACAGCGTCG-3', 5 'Biotin-CAACCAGATATGTAGGCAATGGAATGACGA-3', the probe is Inv-P, and the sequence is 5 '-FITC-TGCTGCTTTCTCTACTTAACAGTGCTAGTTTA [ THF ] GACCTGAATGAATG-C3-spacer-3'.
The invention also relates to a salmonella specificity detection kit, which comprises the forward and reverse primers, the probe and the lateral flow chromatography test strip:
the 5' end of the reverse primer is marked by biotin;
the 5 'end of the probe was labeled with Fluoroescein Isothiocyanate (FITC), the middle region C bases (at least 30bp from the 5' end and at least 15bp from the 3 'end) were replaced with [ THF ], and the 3' end was end-blocked with C3-spacer.
Preferably, the length of the forward and reverse primers is 30 bp.
Preferably, the probe is 48bp in length.
Preferably, the detection kit further comprises one or more of a gel recovery reagent, a nucleic acid extraction reagent, and an RPA amplification reagent.
Preferably, the RPA amplification reagent is a recombinase polymerase isothermal amplification reagent.
The invention has the beneficial effects that:
the method disclosed by the invention can be used for rapidly detecting the food-borne salmonella by combining the RPA with the colloidal gold test strip, can realize real-time, real-time and instant rapid field detection in a non-laboratory environment independent of instruments and equipment, and has the advantages of high accuracy and short time consumption.
Drawings
FIG. 1 is a schematic view of a test strip assembly;
FIG. 2 shows the results of specific screening among probe species in the examples of the present invention;
FIG. 3 is a diagram illustrating the results of the system optimization test in an embodiment of the present invention;
FIG. 4 shows the result of the determination of the lowest limit of detection of RPA-LFS in the example of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
The invention relates to a specific forward and reverse primer and a probe for salmonella, wherein the forward primer and the reverse primer are Inv-F, Inv-R respectively, and the sequences are
5'-CTACAAGCATGAAATGGCAGAACAGCGTCG-3', 5 'Biotin-CAACCAGATATGTAGGCAATGGAATGACGA-3', the probe is Inv-P, and the sequence is 5 '-FITC-TGCTGCTTTCTCTACTTAACAGTGCTAGTTTA [ THF ] GACCTGAATGAATG-C3-spacer-3'. The invention also relates to a salmonella specificity detection kit, which comprises the forward and reverse primers, the probe and the lateral flow chromatography test strip:
the 5' end of the reverse primer is marked by biotin;
the 5 'end of the probe was labeled with Fluoroescein Isothiocyanate (FITC), the middle region C bases (at least 30bp from the 5' end and at least 15bp from the 3 'end) were replaced with [ THF ], and the 3' end was end-blocked with C3-spacer.
Preferably, the length of the forward and reverse primers is 30 bp.
Preferably, the probe is 48bp in length.
Preferably, the detection kit further comprises one or more of a gel recovery reagent, a nucleic acid extraction reagent, and an RPA amplification reagent.
Preferably, the RPA amplification reagent is a recombinase polymerase isothermal amplification reagent.
Examples
Test materials
The genomes of standard strains of salmonella, staphylococcus aureus, enteropathogenic escherichia, listeria monocytogenes and bacillus cereus are provided by food cosmetic quarantine in Wuhan city;
Bas ic kit、
nfokit was purchased from TwistDX, UK; the lateral flow chromatography test strip is purchased from Hangzhou Yosidada biotechnology limited; the PCR cleaning kit is purchased from Moner Biotech Ltd; qubit 4 was purchased from Thermo Scientific; MonAmpTMSYBR Green was purchased from Morina Biotech Ltd; the primers and probes used in the research are synthesized by general biosystems (Anhui) Inc.; roche LightCycler480 fluorescent quantitative PCR instrument purchased from Roche pharmaceutical.
Test method
Primer and probe design
InvA and InvE gene sequences of Salmonella were obtained from NCBI and theoretically feasible specific primers were designed using Primer-Blast with different parameters.
RPA reaction screening primer
Primer screening method
Basic kit reaction system: Inv-F (10. mu.M) 2.1. mu. L, Inv-R (10. mu.M) 2.1. mu.L, 2X Reaction Buffer 25. mu.L, 10X Basic E-mix 5. mu. L, dNTP (1.8mM) 2.25. mu.LL、Template 1μL、ddH2O6.95. mu.L, 20Xcore Reaction mix 2.5. mu. L, MgOAc (280mM), 2.5. mu.L for 50. mu.L Reaction; reaction temperature: 37 ℃; reaction time: 30 min; the sensitivity of the primers was checked by agarose gel electrophoresis and UV gel imager.
RPA-LFS screening probe
Specific probes are designed between forward primers and reverse primers by using Primer Premier 5 software, and the forward Primer dimer, hairpin structure, mismatching, probe and reverse Primer dimer and the like are avoided as much as possible theoretically. Use of probes for screening
nfo kit, reaction system: Inv-F (10. mu.M) 2.1. mu. L, Inv-R (10. mu.M) 2.1. mu. L, Inv-P (10. mu.M) 0.6. mu.L, Rehydration Buffer 29.5. mu. L, ddH2O11.2. mu.L, adding the freeze-dried powder, uniformly mixing, and reacting with 2.5. mu.L of Template 2. mu. L, MgOAc (280mM) for 50. mu.L; reaction temperature: 37 ℃; reaction time: 30 min; and immediately adding EDTA (ethylene diamine tetraacetic acid) or placing the mixture on ice to stop the reaction after the reaction is finished, detecting the reaction by using a lateral flow chromatography test strip to obtain an optimal probe forward and reverse primer, and verifying the result by using agarose gel electrophoresis.
Test paper strip detection principle
And (3) sucking 2 mu L of amplification product which does not need to be purified, adding the amplification product into 98 mu L of buffer solution, inserting a test strip, and allowing the amplification product, the gold-labeled antibody and the buffer solution to flow to the end of the absorbent paper under the siphoning action of the absorbent paper of the test strip. When an amplification product exists, FITC is modified at one end of the amplification product and is combined with a mouse anti-FITC gold-labeled antibody, then the liquid phase continuously flows to the end of the absorbent paper and reaches the detection line, and the amplification product modified with biotin at the other end of the streptavidin fixed on the detection line is captured, so that the colloidal gold combined at one end of the amplification product stays at the detection line, and a red strip is generated. And (4) continuously carrying out chromatography on the redundant colloidal gold, and finally capturing by the Fc fragment antibody of the anti-mouse antibody on the quality control line so as to ensure that the quality control line is developed as well. When the amplification product does not exist, the colloidal gold particles cannot stay at the detection line and are captured by the anti-mouse antibody on the quality control line, so that the color development condition of the quality control line can be used for controlling the quality of the test strip, and the schematic diagram is shown in fig. 1.
Inter-species specificity discrimination
The test strip is used for interspecific specificity screening, meanwhile, salmonella primer probes are used for amplifying salmonella, bacillus cereus, enteropathogenic escherichia, listeria monocytogenes and staphylococcus aureus respectively, and the result is shown in figure 2.
System optimization
And optimizing the screened primer probes, wherein the optimized conditions are reaction time and reaction temperature. The reaction time is respectively set to be 5min, 10min, 10min, 20min, 25min, 30min, 35min and 40 min. The reaction temperatures were set at 22 deg.C, 26 deg.C, 30 deg.C, 34 deg.C, 38 deg.C, 42 deg.C, 46 deg.C. As a result, as shown in FIG. 3, the optimum reaction temperature and time were 42 ℃ and 30 min.
Minimum detection lower limit assay
Since the initial level of bacteria in the diet was not determined, the concentration of template detected during the set-up of the method was generally high under laboratory conditions and was also very sensitive to the presence of very low levels of bacteria in the actual assay, the lowest lower detection limit was determined and the result of using RPA-LFS for Salmonella detection at 10 CFU/. mu.L for this study is shown in FIG. 4.
Comparison with qPCR detection results
At present, qPCR detection is a well-known method which is relatively sensitive and accurate, the qPCR detection result is used as a control, the accuracy of the RPA-LFS for detection can be demonstrated, the RPA-LFS method can be used for realizing real-time, real-time and instant rapid field detection under the condition of the same detection accuracy without depending on instruments and equipment, 50 samples of artificially polluted fresh milk are simultaneously detected by respectively using the RPA-LFS and the qPCR, 8 positive results are detected totally, the results of the two methods are consistent, and the results are shown in a table.
(“+”means positive.“-”means negative.)
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (6)
1. The specific forward and reverse primers and probes for salmonella are characterized in that the forward primer and the reverse primer are Inv-F, Inv-R respectively and have sequences of CTACAAGCATGAAATGGCAGAACAGCGTCG, Biotin-CAACCAGATATGTAGGCAATGGAATGACGA respectively, the probe is Inv-P and has a sequence of FITC-TGCTGCTTTCTCTACTTAACAGTGCTAGTTTA [ THF ]
GACCTGAATGAATGA-C3 Spacer。
2. A salmonella-specific detection kit comprising the forward and reverse primers and probe of claim 1, and a lateral flow chromatography test strip:
the 5' end of the reverse primer is marked by biotin;
the 5 'end of the probe was labeled with Fluoroescein Isothiocyanate (FITC), the middle region C bases (at least 30bp from the 5' end and at least 15bp from the 3 'end) were replaced with [ THF ], and the 3' end was end-blocked with C3-spacer.
3. The detection kit according to claim 2, wherein the length of the forward and reverse primers is 30 bp.
4. The detection kit according to claim 2, wherein the probe is 48bp in length.
5. The detection kit according to any one of claims 2 to 4, wherein the detection kit further comprises one or more of a gel recovery reagent, a nucleic acid extraction reagent, and an RPA amplification reagent.
6. The detection kit according to claim 5, wherein the RPA amplification reagent is a recombinase polymerase isothermal amplification reagent.
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Cited By (2)
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| CN113528613A (en) * | 2021-07-16 | 2021-10-22 | 安徽农业大学 | Experimental method for detecting bacterial genes based on multienzyme constant-temperature nucleic acid rapid amplification technology |
| CN113832241A (en) * | 2021-08-27 | 2021-12-24 | 江苏海洋大学 | Probe, primer group, kit and detection method for rapidly detecting aeromonas salmonicida through RPA-LFS |
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Cited By (2)
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| CN113832241A (en) * | 2021-08-27 | 2021-12-24 | 江苏海洋大学 | Probe, primer group, kit and detection method for rapidly detecting aeromonas salmonicida through RPA-LFS |
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