CN111621578A - RU 61-00441 gene-based Salmonella spelt constant-temperature detection kit and method - Google Patents
RU 61-00441 gene-based Salmonella spelt constant-temperature detection kit and method Download PDFInfo
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- CN111621578A CN111621578A CN202010414399.3A CN202010414399A CN111621578A CN 111621578 A CN111621578 A CN 111621578A CN 202010414399 A CN202010414399 A CN 202010414399A CN 111621578 A CN111621578 A CN 111621578A
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides an RPA primer for isothermal detection of Salmonella spelt and a detection method. The primer sequences are as follows: for the RU61 — 00441 gene: the upstream primer is SEQ ID NO. 1: 5'-GACCAGTTCTTCTTCATGACCTACG AAATC-3', respectively; the downstream primer is SEQ ID NO. 2: 5'-TTAATCCACACCTTTGGCGGAA TTAGCTTA-3' are provided. The invention realizes the rapid and accurate detection of the salmonella spelt based on the recombinase polymerase amplification technology. The detection method provided by the invention comprises the following steps: (1) extracting sample DNA, RPA amplifying (2) detecting amplified product by gel electrophoresis (3) comparing bands after electrophoresis, if there is a band at 200bp position, it is proved that the sample contains Salmonella delphinii. The kit and the method can specifically distinguish serotype salmonella delphinium and other serotypes of salmonella, and are suitable for field detection of enterprises.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a RU 61-00441 gene-based Salmonella spelt constant-temperature detection kit and a method.
Background
Salmonella is a common food-borne pathogenic bacterium. The traditional method for identifying salmonella is mainly based on morphological characteristics, culture characteristics, physiological and biochemical characteristics, antigen characteristics, bacteriophage characteristics and the like. Salmonella spelt can cause diseases of livestock and poultry and can also cause food poisoning of people. The detection of the pathogenic bacteria is an important link for detecting the pathogenic bacteria, and has important significance in occasions such as public health and the like.
The existing salmonella spelts detection method needs serotype experiments, has long detection time and expensive equipment, and is not convenient to popularize and use in the basic level.
Disclosure of Invention
The invention aims to obtain a specific detection target gene of the salmonella delphinii through genome comparative analysis, so that the target gene is subjected to constant-temperature RPA amplification and electrophoresis detection through a specific primer pair, the problems of inaccurate detection result, complex detection process and long detection period are solved, and the purpose of high specificity, rapidness and on-site detection of the salmonella delphinii is realized.
An RPA primer pair for detecting salmonella spelts, characterized in that it gives rise to the following sequences: for the RU 61-00441 gene,
the upstream primer is SEQ ID NO. 1: 5'-GACCAGTTCTTCTTCATGACCTACGAAATC-3'
The downstream primer is SEQ ID NO. 2: 5'-TTAATCCACACCTTTGGCGGAATTAGCTTA-3' are provided.
The application of the RPA primer for detecting the salmonella spelts in the detection of the salmonella spelts is characterized in that the method for detecting the salmonella spelts by using the RPA primer comprises the following steps:
(1) extracting sample DNA, and amplifying RPA; (2) detecting the amplification product by gel electrophoresis; (3) comparing the bands after electrophoresis, if a band exists at the position of 200bp, the sample is proved to contain the Salmonella delavata.
The application of the RPA primer for detecting the salmonella spelts in detecting the salmonella spelts is characterized in that the method for detecting the salmonella spelts by using the RPA primer comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) combining the sequences of claim 1 into an RPA-specific primer, performing an RPA amplification reaction;
(3) and (3) judging an amplification result by adopting agarose gel electrophoresis under an ultraviolet lamp, comparing bands after electrophoresis, and if a band exists at a position of 200bp, proving that the sample contains the salmonella spelt.
The application of the RPA primer for detecting the salmonella spelts in detecting the salmonella spelts is characterized in that:
wherein 50ul of the RPA amplification reaction detection system comprises: 1 Xreaction buffer 44ul, 10uM primer 2ul each, template 2 ul.
The application of the RPA primer for detecting the salmonella spelts in detecting the salmonella spelts is characterized in that the RPA amplification reaction program parameters are as follows: at 37 deg.C, reacting for 25-30min, and performing agarose gel electrophoresis.
A kit for detecting salmonella spelts by using RPA primers for detecting salmonella spelts is characterized in that: comprising the pair of RPA primers of claim 1. The invention provides a method for detecting salmonella spelt, wherein a detection result of a DNA fragment separated from a 200bp position after electrophoresis is positive.
The invention provides a method for detecting salmonella spelt, wherein 50ul of an RPA detection system comprises the following components: 1 Xreaction buffer 44ul, 10uM primer 2ul each, template 2 ul.
The invention provides a method for detecting salmonella spelt, wherein the reaction program of RPA comprises the following steps: at 37 deg.C, reacting for 25-30min, and performing agarose gel electrophoresis.
The method for detecting the salmonella spelt provided by the invention is measured by a 50ul PCR reaction system, and the total DNA concentration of the salmonella spelt is required to be more than or equal to 76.12 fg/ul.
Compared with the prior art, the invention has the beneficial effects that:
(1) the detection method can directly identify the salmonella spelt, does not need serotype experiments, shortens the detection time within 6h, reduces the detection cost and accelerates the detection speed.
(2) The invention has the advantages that the detection sensitivity is higher than that of the common PCR detection by the RPA amplification technology, the detection is more accurate, and the false negative result interference of low-pollution measurement is avoided.
(3) The invention adopts the constant temperature amplification technology, avoids the investment of expensive equipment with variable temperature and is convenient for popularization and use at the basic level.
Drawings
FIG. 1 sensitivity experiment for RPA detection of DNA
M: DL 2000; template concentration: 1-8: 76.12ng/ul, 7.612ng/ul, 761.2pg/ul, 76.12pg/ul, 7.612pg/ul, 761.2fg/ul, 76.12fg/ul and a positive control
FIG. 2RPA test for colony sensitivity
M is DL2000, the template concentration is 1-7: 4.5 × 107CFU/mL、4.5×106CFU/mL、4.5×105CFU/mL、4.5×104CFU/mL、4.5×103CFU/mL、4.5×102CFU/mL and 4.5 × 101CFU/mL
Detailed Description
The present invention is further illustrated by the following examples, in which experimental methods not specifying specific conditions are generally performed according to methods known in the art or according to manufacturer's recommendations, and the strains involved in the examples are known in the art and can be readily obtained from public commercial sources by those skilled in the art.
Selection of serotype specific target gene and design of RPA primer
1. Screening of salmonella spelt serotype specific gene
The whole genome sequence of the salmonella delustris CVM40386 strain is obtained in an NCBI database, each gene of the genome of the strain is compared by using a Blast program of an NCBA website according to a method for comparing the genome, and a gene which has higher homology (E value is 0 and Query cover is 0%) with the serotype and has low homology (Quercover is less than 10%) with other microorganisms is selected as a quasi-specific gene of the salmonella paratyphi. By the method, 7 quasi-specific genes of the salmonella paratyphi C are obtained, a plurality of pairs of primers are designed for each gene, and specificity verification is performed by using other serotypes of the salmonella stored in a laboratory. According to the result of specificity verification, the RU 61-00441 gene is determined to be used as a specific detection target of the salmonella spelt serotype.
2. Primer design
According to the requirement of RPA on primer design, designing a primer by Primer5.0 software, setting the GC% range to be 40-60%, setting the product size range to be 200-600bp, selecting a primer from an alternative primer pair, verifying the specificity, screening to obtain primers which are respectively shown as (SEQ ID NO.1 and SEQ ID NO.2) and entrusted to Shanghai Bioengineering technology service Limited company for synthesis.
The upstream primer is SEQ ID NO. 1: 5'-GACCAGTTCTTCTTCATGACCTACGAAATC-3'
The downstream primer is SEQ ID NO. 2: 5'-TTAATCCACACCTTTGGCGGAATTAGCTTA-3'
Establishment of Salmonella delavata RPA constant-temperature amplification detection method
Preparation of DNA template
The serotypes including 45 salmonella serpyllum standard strains such as salmonella spelt (S.Derby) and 18 non-salmonella (shown in table 1) (the strain with the number of CICC is purchased from China Industrial microbiological culture Collection center, the strain with the number of CMCC is purchased from China medical microbiological culture Collection center, the unnumbered strain is self-preserved in a laboratory, and if other colleagues need research, the laboratory is willing to provide the strain) are respectively inoculated into 30ml of LB liquid culture medium and cultured for 8 hours at 37 ℃. Respectively taking 1ml of each bacterial suspension in a 1.5ml centrifuge tube, carrying out centrifugation at 12000r/min for 1min, and discarding the supernatant. The pellet was washed twice with 500ul of sterilized pure water, and the pellet was dissolved in 100ul of sterilized pure water. Boiling in boiling water bath for 10min, 12000r/min, centrifuging for 1min, and taking supernatant as PCR template.
Establishment of RPA reaction System
Establishing an RPA detection system: 1 Xreaction buffer 44ul, 10uM primer 2ul each, template 2 ul.
Design of the RPA reaction program: the reaction time is 25-30min at 37 ℃.
And (3) respectively carrying out RPA amplification by using the DNA of each bacterial strain extracted in the steps as a template in a constant-temperature metal bath instrument according to a set reaction system and a set reaction program, and carrying out gel electrophoresis for detection.
5. Gel electrophoresis detection
The PCR-amplified products, DNALader I and ddH2O as markers were added to 1% agarose gel in an order of 3ul, followed by 100V for 40min, and then EB-staining was performed.
According to the RPA gel detection result, the salmonella DNA including the salmonella spelt is used as a template, and only the salmonella spelt appears a specific electrophoresis band in 200bp after RPA amplification; to better illustrate the specificity of the present invention, a total of 45 and 18 non-salmonella (where "+" represents isolated strain and others are standard strains) from different sources of salmonella were subjected to RPA amplification and gel electrophoresis results are shown in table 1. When an amplification band appears at a position of 200bp in the electrophoresis result, the result is proved to be positive and is indicated by "+"; when the position has no amplified band, a negative result is proved, and is indicated by "-"; as can also be seen from Table 1, only Salmonella spelt showed a positive reaction by RPA amplification by this method. The result well illustrates that the method provided by the invention has high stability, strong specificity, wide application range and accurate detection result.
Third, sensitivity evaluation of RPA detection method
Evaluation of template sensitivity of RPA detection method
The screening of the salmonella speziliensis serotype specific gene and the primer design step are as the first step.
Preparation of DNA template: the single colony of the salmonella paratyphi C is picked and transferred into 30ml LB liquid culture medium, and cultured overnight at 37 ℃. The total DNA of Salmonella delavata is extracted by the small extraction kit of bacterial genome DNA produced by the marine bioengineering technology service company Limited, and the concentration is 76.12ng/ul by determination. Sterile water was used for 10-fold gradient dilution for a total of 7 gradients as RPA template.
Establishing an RPA reaction system: establishing an RPA detection system: 50ul contains: 1 Xreaction buffer 44ul, 10uM primer 2ul each, template 2 ul. Reaction procedure for RPA: at 37 deg.C, reacting for 25-30min, and performing agarose gel electrophoresis.
And (3) taking 2ul of each of the 7 gradient DNA templates in the step, adding the DNA templates into an RPA reaction system, and carrying out reaction amplification on a metal bath. 5ul of the RPA amplification product was applied to a 1% agarose gel and observed after EB staining after 40min at 100V.
The band corresponding to 200bp in lanes 1 to 6 shows that the concentration in lane 7 is 7.612fg/ul, and the electrophoresis result is shown in FIG. 1. Therefore, the sensitivity of DNA detection to the RPA primer was determined to be 76.12 fg/ul.
Evaluation of colony sensitivity in RPA detection method
The screening of the salmonella speziliensis serotype specific gene and the primer design step are as the first step.
Preparing DNA template, selecting single colony of Salmonella paratyphi C.and transferring it into 30ml LB liquid culture medium, overnight culture at 37 deg.C, using sterile physiological saline to make 10-fold gradient dilution, making total dilution with 7 gradients, using thermal cracking method to extract total DNA of Salmonella paraphilia at every dilution, and measuring that initial concentration of bacterial liquid is 4.5 × 107CFU/mL。
Establishing an RPA reaction system: establishing an RPA detection system: 50ul contains: 1 Xreaction buffer 44ul, 10uM primer 2ul each, template 2 ul. Reaction procedure for RPA: at 37 deg.C, reacting for 25-30min, and performing agarose gel electrophoresis.
And (3) taking 2ul of each of the 7 gradient DNA templates in the step, adding the DNA templates into an RPA reaction system, and carrying out reaction amplification on a metal bath. 5ul of the RPA amplification product was applied to a 1% agarose gel and observed after EB staining after 40min at 100V.
The band corresponding to 200bp of the RPA primer pair from lane 1 to lane 6 shows that the concentration of the bacterial solution in lane 7 is 45CFU/mL, and the electrophoresis result is shown in FIG. 2. Therefore, the colony sensitivity for RPA primer detection was determined to be 450 CFU/mL.
Table 1: strain for evaluating specificity and detection result
It will be understood that the above-described embodiments are merely illustrative of the principles of the invention, which is not limited thereto, and that various modifications and changes can be made by those skilled in the art without departing from the spirit of the invention, which also falls within the scope of the invention.
Claims (6)
1. An RPA primer pair for detecting salmonella spelts, characterized in that it gives rise to the following sequences: for the RU 61-00441 gene,
the upstream primer is SEQ ID NO. 1: 5'-GACCAGTTCTTCTTCATGACCTACGAAATC-3'
The downstream primer is SEQ ID NO. 2: 5'-TTAATCCACACCTTTGGCGGAATTAGCTTA-3' are provided.
2. The application of the RPA primer for detecting the salmonella spelts in the detection of the salmonella spelts is characterized in that the method for detecting the salmonella spelts by using the RPA primer comprises the following steps:
(1) extracting sample DNA, and amplifying RPA; (2) detecting the amplification product by gel electrophoresis; (3) comparing the bands after electrophoresis, if a band exists at the position of 200bp, the sample is proved to contain the Salmonella delavata.
3. The use of the RPA primers for detecting salmonella spelt of claim 2 for detecting salmonella spelt, wherein the method for detecting salmonella spelt using the RPA primers comprises the steps of:
(1) extracting DNA of a sample to be detected;
(2) combining the sequences of claim 1 into an RPA-specific primer, performing an RPA amplification reaction;
(3) and (3) judging an amplification result by adopting agarose gel electrophoresis under an ultraviolet lamp, comparing bands after electrophoresis, and if a band exists at a position of 200bp, proving that the sample contains the salmonella spelt.
4. The use of the RPA primers for detecting salmonella spelt of claim 3 for detecting salmonella spelt, wherein:
wherein 50ul of the RPA amplification reaction detection system comprises: 1 Xreaction buffer 44ul, 10uM primer 2ul each, template 2 ul.
5. The use of the RPA primers for detecting salmonella spelts of claim 4 for detecting salmonella spelts, wherein the RPA amplification reaction program parameters are: at 37 deg.C, reacting for 25-30min, and performing agarose gel electrophoresis.
6. A kit for detecting salmonella spelts by using RPA primers for detecting salmonella spelts is characterized in that: comprising the pair of RPA primers of claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023163458A1 (en) * | 2022-02-23 | 2023-08-31 | 주식회사 풀무원 | Crispr-cas-based composition for salmonella detection and salmonella detection method using same |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023163458A1 (en) * | 2022-02-23 | 2023-08-31 | 주식회사 풀무원 | Crispr-cas-based composition for salmonella detection and salmonella detection method using same |
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