CN112980978A - Rapid detection method for bacillus cereus based on RPA-LF - Google Patents
Rapid detection method for bacillus cereus based on RPA-LF Download PDFInfo
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- CN112980978A CN112980978A CN202110391994.4A CN202110391994A CN112980978A CN 112980978 A CN112980978 A CN 112980978A CN 202110391994 A CN202110391994 A CN 202110391994A CN 112980978 A CN112980978 A CN 112980978A
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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 pathogenic bacteria detection, and discloses a rapid detection method for bacillus cereus based on RPA-LF, which is characterized by comprising the following steps: taking a bacillus cereus conserved virulence gene Nhe as a target sequence to design a primer and a probe, wherein an upstream primer 5 '-CTAGTTATGAGACATGGTTAAAATCCACAACGGC-3' thereof and a downstream primer: 5 '-Biton-CCCAAAGTCAAGCTGAAGAGGACA TTGATCTGCC-3' combines the RPA technology and the LF technology on the basis, and a method for rapidly detecting the bacillus cereus on site through the RPA-LF technology is constructed.
Description
Technical Field
The invention relates to the fields of food safety and livestock breeding, in particular to a rapid detection method for bacillus cereus based on RPA-LF, and also relates to an application of RPA-LF in bacillus cereus detection.
Background
Bacillus cereus (bc) is a gram-positive Bacillus of Bacillus, which is a short-chain or long-chain gram-positive Bacillus, exists in soil, dust, sewage, feed, plants and various cooked foods, and has strong resistance to conditions such as high temperature, ultraviolet rays, electromagnetic radiation, harmful chemical substance stimulation and the like. The bacteria cause food poisoning of people most commonly, and can cause mastitis and endometritis of milk cows, diarrhea of dogs and cats, death of tilapia and the like besides infecting people, thereby bringing serious harm to food safety and the development of the breeding industry.
The bacillus cereus specified by national standard GB 4789.14-2014 is mainly detected by a traditional culture method, namely, a sample to be detected is subjected to separation culture after bacteria are enriched, then, morphological and biochemical identification is carried out on a suspicious strain subjected to separation culture, and then, comprehensive evaluation is carried out on an identification result to determine whether the suspicious strain is the bacillus cereus. The method has long detection period, complicated process and high requirement on the experimental skill of workers, and can not be used for field detection only in laboratory operation, so that the method is not beneficial to the rapid detection of the bacillus cereus, and the detection requirement of the bacillus cereus in economic laggard areas can not be met.
The Recombinase Polymerase Amplification (RPA) is a novel constant temperature Amplification technology, and the technology mainly depends on three substances of Recombinase, single-stranded DNA binding protein and strand displacement DNA Polymerase, so that the exponential Amplification of nucleic acid can be realized under the constant temperature condition, and the RPA detection method with sensitive and rapid reaction is formed. In 2014, the RPA technology is used for Lateral Flow (LF) detection, and an RPA-LF system is established. After the system is subjected to constant-temperature amplification reaction for a short time, the detection result can be observed by naked eyes, a valuable instrument is not needed, the operation is simple, and the system is suitable for rapid field detection.
However, it is difficult to implement this technique because it requires the design of specific primers and probes for the microorganism to be tested, and a series of specific methods and technical parameters are used. Thus, there is no report of the use of RPA-LF for the detection of Bacillus cereus, particularly, bovine pathogenic Bacillus cereus.
Disclosure of Invention
The invention aims to provide a rapid detection method which can carry out on-site rapid detection on bacillus cereus, and has the advantages of simple operation and low cost.
In order to achieve the purpose, the invention adopts the following technical scheme:
the rapid detection method for the bacillus cereus based on the RPA-LF is provided, and is characterized by comprising the following steps: primers and probes are designed by taking a bacillus cereus conserved virulence gene Nhe as a target sequence: the length of the primer reaches 30-35 bp, the GC content accounts for 40% -60%, the primer does not contain a repetitive sequence, the length of an amplified fragment does not exceed 500 bp, and the 5' -end of the downstream primer is labeled with biotin; the length of the probe is at least 42 bp, the 5 'end is labeled by a fluorescent group (FAM), the 3' end is introduced with a polymerase amplification reaction interrupting group (3C-spacer) for end blocking, THF is introduced in the middle of the probe, namely a purine-free pyrimidine-free site (AP site) is not included, at least 25 bp of bases are reserved before the AP site, and at least 15bp of bases are reserved after the AP site.
The length of the primer amplification segment is 100-200 bp.
The primers and probes are designed as follows:
(1) primer:
an upstream primer: 5' -CTAGTTATGAGACATGGTTAAAATCCACAACGGC-3
A downstream primer: 5 '-Biton-CCCAAAGTCAAGCTGAAGAGGACATTGATCTGCC-3' -shaped
(2) And (3) probe:
FAM-CATAACTTGAAGAAAGACGTATTAACGATT(THF)ACAGCTCCGAATGAAT-C3 Spacers
the detection temperature of the bacillus cereus is 36-40 ℃.
The detection time of the bacillus cereus is less than or equal to 15 min.
The above-mentionedThe detection concentration of Bacillus cereus has a lower limit of 1.0 × 102 CFU·mL-1。
Has the advantages that: the RPA technology and the LF technology are combined, the method for rapidly detecting the bacillus cereus on site through the RPA-LF is constructed, instrument facilities in a laboratory are not needed, high-quality technicians are not relied on, and farmers can complete detection of the bacillus cereus through color reaction in a farm, so that conditions are provided for preventing and treating the bacillus cereus disease, and particularly preventing the bacillus cereus from damaging cattle.
Drawings
FIG. 1 is a test strip morphology chart for specific detection of RPA-LF;
FIG. 2 is a test strip morphology for the sensitivity detection of RPA-LF.
Detailed Description
A method for rapidly detecting Bacillus cereus RPA-LF mainly comprises the following steps:
1. designing and screening primers:
during design, the length of the primer is up to 30-35 bp, the GC content is 40% -60%, the primer does not contain a repetitive sequence, the length of an amplified fragment is not more than 500 bp, wherein 100-200 bp is used as the optimal amplification length, and the 5' end of a downstream primer is labeled with biotin;
the specific method comprises the following steps: taking a conserved sequence of a bacillus cereus nonhemolytic enterotoxin gene JQ039111 published by GenBank as a template, using NCBI primer-blast to design a primer, using the virulence gene DNA as the template to perform RPA amplification, using 1.5% agarose gel electrophoresis to detect an amplification product, and screening out a primer with the best amplification effect. The RPA-LF primer needs to be labeled with Biotin (Biotin) at the 5' end. The following primers can be designed:
an upstream primer: 5' -CTAGTTATGAGACATGGTTAAAATCCACAACGGC-3
A downstream primer: 5 '-Biton-CCCAAAGTCAAGCTGAAGAGGACATTGATCTGCC-3' -shaped
2. Designing and screening a probe:
and designing a probe by using primer 5.0 software according to the primer with the best amplification effect and better interspecies specificity, wherein the probe is positioned between the upstream primer and the downstream primer. The design principle of the probe is as follows:
(1) the length of the probe is at least 46 bp;
(2) labeling the 5 'end with carboxyfluorescein (FAM), introducing 3C-spacer into the 3' end for end blocking, and introducing tetrahydrofuran abasic site (THF), i.e., purine-free and pyrimidine-free site (AP site), into the middle of the probe;
(3) at least 30 bp of bases are reserved before the AP site, at least 15bp of bases are reserved after the AP site, and biotin is labeled at the 5' end of the downstream primer. In this way, the probe and the downstream primer amplification product carry FAM at one end and Biotin (Biotin) at the other end, and thus can be detected by LF. The designed probe is sent to the biological engineering company with the limited responsibility for synthesis, and the sequence of the probe is as follows.
FAM-CATAACTTGAAGAAAGACGTATTAACGATT(THF)ACAGCTCCGAATGAAT-C3 Spacers。
Through the process, an nfo probe containing a THF (tetrahydrofuran) site and a fluorescent group (FAM) is designed, and simultaneously, the primer is labeled by Biotin (Biotin), so that the double-labeled amplicon with both a probe fluorescent group label and a primer label is obtained. The double-labeled amplicon can be used for preparing a lateral flow chromatography test strip. The specific method comprises the following steps:
firstly preparing immune colloidal gold solution and identifying the quality of the immune colloidal gold solution, then evenly coating the prepared colloidal gold solution on a gold label pad at 30 mL/cm, and placing the gold label pad in a vacuum drying oven at 37 ℃ for overnight. Secondly, secondary antibodies of streptavidin and anti-carboxyfluorescein antibody (FAM) are sprayed on the NC membrane at 1 pL/cm as a detection line (T line) and a quality control line (C line), and are dried at 37 ℃ overnight. And finally, sequentially connecting the sample pad, the combination pad, the NC membrane, the back plate and the absorbent paper in an overlapping manner from left to right, assembling the five parts, cutting the five parts into test strips of 6 cm multiplied by 4 mm, sealing and storing the test strips, and preparing the lateral laminar flow test strips sold on the market. In order to facilitate the application and improve the detection accuracy, the test strip can be obtained by direct purchase. Wherein the sample pad comprises colloidal gold particles labeled with FAM antibody and a function of filtering a specific sample component, and the conjugate pad can serve as a label-attached carrier. When the detection result is positive, the FAM antibody coupled with the colloidal gold particles and the streptavidin on the T line are respectively combined with the FAM antibody coupled with the colloidal gold particles and the streptavidin on the T line in the lateral flow chromatography process, so that the T line is developed, and meanwhile, the FAM antibody coupled with the colloidal gold particles is combined with the anti-FAM secondary antibody at the C line, so that the C line is developed. If the result is negative, only the C line develops color, and the T line does not develop color. The RPA-LF has the advantage that the detection result can be observed by naked eyes after a short-time constant-temperature amplification reaction.
In order to prove the effect of the technical scheme, the optimal using method is searched, and a large number of experiments are carried out. The following illustrates only one representative experiment:
(1) strains and experimental animals: bacillus cereus is from the spleen of a sudden death cow in a cattle farm; clostridium perfringens, Bacillus megaterium and Bacillus subtilis are all provided by veterinary pathology laboratories of the institute of animal science and technology of the university of inner Mongolia.
(2) Main reagents and instruments: sodium chloride, nutrient agar, glucose, etc. were purchased from Beijing chemical plants; yeast extract and peptone were supplied by the company obozin; the bacterial genome DNA extraction kit is purchased from TaKaRa company, an electric heating constant temperature incubator, a constant temperature water bath, a 5424R desk-top high-speed centrifuge, a full-automatic gel imaging analysis system ZF-258, an electrophoresis apparatus, an electronic balance, an ultra-clean workbench and the like.
(3) Determination of specificity of RPA-LF
Extracting the bacterial genome DNA according to the DNA extraction specification, respectively extracting the DNAs of clostridium perfringens, bacillus megaterium and bacillus subtilis 3 bacillus as templates, and setting a physiological saline negative control group for RPA-LF detection. The result after the reaction at 38 ℃ for 15min is shown in figure 1, test paper in the figure sequentially comprises a normal saline control group, a clostridium perfringens group, a bacillus megaterium group, a bacillus subtilis group and a bacillus cereus group from left to right, a clear detection strip appears on the test paper of the bacillus cereus detection group, the detection result is positive, the other test groups and the normal saline negative control group have the same result, no detection strip appears, and the result is negative. The test result shows that the detection method has higher specificity and also shows that the specificity between the primers and the probes is better, so that the pair of primers and the probes can be selected to optimize the reaction conditions.
(4) RPA-LF reaction temperature optimization
Taking a conserved sequence of a non-hemolytic enterotoxin gene JQ039111 of bacillus cereus as a template, and setting different temperature gradients after amplification by using the screened optimal primers and probes: 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 8 different temperature test groups. After the addition according to the table 1, the reaction is carried out for 20 min at the constant temperature, and after the reaction is finished, the lateral cross flow test strip is used for detection, and the optimal reaction temperature area is selected. Then, each temperature in the area was tested with 2 ℃ as the group interval, and the test results were recorded and analyzed.
The RPA reaction product is added to lateral cross flow test strips for 5min, and is visible, no strip exists in test strip detection lines at 10 ℃, 15 ℃, 20 ℃, 25 ℃ and 45 ℃, light strips appear in test strips at 30 ℃, and obvious in test strips at 35 ℃ and 40 ℃. After 10 min, no strip appears in the test strip detection lines at 10 ℃, 15 ℃ and 20 ℃, light strips appear in the test strip detection lines at 25 ℃ and 45 ℃, and the test strip detection lines at 30 ℃, 35 ℃ and 40 ℃ are obvious. The result shows that the temperature is 35-40 ℃ which is the optimal reaction temperature range of the RPA in the group. In order to narrow the optimal reaction temperature range and determine the optimal reaction temperature, the reaction temperature is set to be 34 ℃, 36 ℃, 38 ℃ and 40 ℃ according to the temperature range of 35-40 ℃ for detection. The result shows that the obvious band appears more rapidly in the group of 36 ℃, 38 ℃ and 40 ℃ than in the group of 34 ℃, which indicates that the optimal temperature interval of the reaction is 36-40 ℃. In this experiment 38 ℃ was set as the reaction temperature.
(5) RPA-LF reaction time optimization
And (3) taking the conserved sequence of the non-hemolytic enterotoxin gene JQ039111 of the bacillus cereus as a template, and amplifying by using the screened optimal primer and probe. In order to find out the optimal reaction time of the RPA experiment, 5min, 10 min, 15min, 20 min, 25 min and 30 min are set, and 6 different reaction time test groups are set. After the addition according to table 1, the reaction was carried out at a constant temperature of 38 ℃, and after the reaction was completed, the lateral cross-flow test strip was used for detection, and the region with the best reaction time was selected. The RPA reaction product under each reaction duration is added to the lateral cross flow test strip for 15min, so that no strip appears in a 5min reaction group, detection strips appear in both the 10 min and 15min reaction group detection test strips, but the detection strips in the 10 min group are shallow, and the detection strips in the other groups are very obvious. The result shows that the reaction time of 10-15 min is the lowest effective detection area of the test.
To determine the shortest effective reaction time for the reaction, the optimal reaction time in the region was determined with 2 min as the intergroup distance. The RPA reaction time is set to be 12 min, 14 min and 16 min, detection strips can be seen in each test group through lateral cross flow test strip detection, but the appearance time and the definition of the strips in the groups of 14 min and 16 min are higher than those in the group of 10 min, so that 15min is set as the optimal reaction time of the reaction in the experiment.
(6) Determination of RPA-LF sensitivity
Counting the number of bacteria in the cultured bacterial liquid for 12 h by using a microscope, calculating the concentration, and adjusting the concentration of the bacterial liquid to 1.0 × 1010 CFU·mL-1As initial concentration, the bacterial liquid concentration is diluted to 1.0 × 10 by multiple dilution9 CFU·mL-1、1.0×108 CFU·mL-1、1.0×107 CFU·mL-1、1.0×106 CFU·mL-1、1.0×105 CFU·mL-1、1.0×104 CFU·mL-1、1.0×103 CFU·mL-1、1.0×102 CFU·mL-1、1.0×101 CFU·mL-1. Extracting DNA of the bacterial liquid with each concentration by using the same bacterial genome DNA extraction method, adding the bacterial liquid according to the RPA-LF reaction components, carrying out vortex oscillation and uniform mixing, and reacting for 20 min at the constant temperature of 38 ℃. Finally, 2 mu L of RPA product is added into 98 mu L of buffer solution and mixed evenly, 10 mu L of mixed solution is dripped into the lateral transverse flow test strip, the reaction time is set to be 15min, the reaction temperature is set to be 38 ℃, and the experiment is recordedAs a result, the sensitivity of the reaction was analyzed.
The reaction results are shown in FIG. 2, in which the test paper is sequentially a normal saline control group with a concentration of 1.0 × 10 from left to right1 CFU·mL-1、1.0×102 CFU·mL-1、1.0×103 CFU·mL-1、1.0×104 CFU·mL-1、1.0×105 CFU·mL-1、1.0×106 CFU·mL-1、1.0×107 CFU·mL-1And (5) detecting the group. As a result, the physiological saline control group was compared with 1.0X 101 CFU·mL-1The test paper of (1.0X 10) shows no detection line2 CFU·mL-1Detection line with group not visible, 1.0X 103 CFU·mL-1Clear detection lines can be seen in the groups, and clear detection lines can be seen in the rest concentration detection groups. The results show that the test can be used for the concentration of 1.0 multiplied by 102 CFU·mL-1 The above bacteria were detected.
In the invention, a portable and rapid bacillus cereus nucleic acid detection method is established based on a recombinant polymerase isothermal amplification technology (RPA) and a lateral transverse flow test strip technology (LF), and the method provides a reference basis for livestock breeding. The invention designs a primer by taking a virulence gene Nhe of bacillus cereus as a target sequence, and simultaneously evaluates the detection time, temperature, specificity and sensitivity of the method by screening out the optimal primer and probe to establish the RPA-LF rapid detection method. The result shows that the method can obtain the detection result within 15min at 36-40 ℃, has no cross reaction with other common bacillus, has high specificity and can reach the concentration of 1.0 multiplied by 102 CFU·mL-1The above bacteria are detected with high sensitivity.
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 are equivalent to or changed within the technical scope of the present invention.
Claims (6)
1. A rapid detection method for Bacillus cereus based on RPA-LF is characterized in that primers and probes are designed by taking a Bacillus cereus conserved virulence gene Nhe as a target sequence: the length of the primer reaches 30-35 bp, the GC content accounts for 40% -60%, the primer does not contain a repetitive sequence, the length of an amplified fragment does not exceed 500 bp, and the 5' -end of the downstream primer is labeled with biotin; the length of the probe is at least 42 bp, the 5 'end is labeled by a fluorescent group (FAM), the 3' end is introduced with a polymerase amplification reaction interrupting group (3C-spacer) for end blocking, THF is introduced in the middle of the probe, namely a purine-free pyrimidine-free site (AP site) is not included, at least 25 bp of bases are reserved before the AP site, and at least 15bp of bases are reserved after the AP site.
2. The rapid detection method for bacillus cereus based on RPA-LF as claimed in claim 1, wherein: the length of the primer amplification segment is 100-200 bp.
3. The rapid detection method for bacillus cereus based on RPA-LF as claimed in claim 1, wherein: the following primers and probes are designed by taking the bacillus cereus conserved virulence gene Nhe as a target sequence:
(1) primer:
an upstream primer: 5' -CTAGTTATGAGACATGGTTAAAATCCACAACGGC-3
A downstream primer: 5 '-Biton-CCCAAAGTCAAGCTGAAGAGGACATTGATCTGCC-3' -shaped
(2) And (3) probe:
FAM-CATAACTTGAAGAAAGACGTATTAACGATT(THF)ACAGCTCCGAATGAAT-C3 Spacers。
4. the rapid detection method for bacillus cereus based on RPA-LF as claimed in claim 2, wherein: the detection temperature of the bacillus cereus is 36-40 ℃.
5. The rapid detection method for bacillus cereus based on RPA-LF as claimed in claim 2, wherein: the detection time of the bacillus cereus is less than or equal to 15 min.
6. The rapid detection method for bacillus cereus based on RPA-LF in claim 3, which comprises the following steps: the detection concentration bottom limit value of the bacillus cereus is 1.0 multiplied by 102 CFU·mL-1。
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Citations (2)
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| WO2011142119A1 (en) * | 2010-05-12 | 2011-11-17 | 東洋製罐株式会社 | Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria |
| CN106434902A (en) * | 2016-08-31 | 2017-02-22 | 北京卓诚惠生生物科技股份有限公司 | Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof |
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Patent Citations (2)
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| WO2011142119A1 (en) * | 2010-05-12 | 2011-11-17 | 東洋製罐株式会社 | Food-poisoning bacteria detection carrier, and method for detecting food-poisoning bacteria |
| CN106434902A (en) * | 2016-08-31 | 2017-02-22 | 北京卓诚惠生生物科技股份有限公司 | Primer set and kit for detecting bacillus cereus virulence genes by means of multiplex-PCR and detection method thereof |
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| LEI WANG等: "A Visualized Isothermal Amplification Method for Rapid and Specific Detection of Emetic and Non-emetic Bacillus cereus in Dairy Products", 《FRONT MICROBIOL》 * |
| 宋丽丽: "蜡样芽孢杆菌的分离鉴定及NHE基因RPA-LF检测方法的建立", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
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