CN107236812B - Detection kit for bacillus cereus, detection method and application - Google Patents
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Abstract
The invention relates to the technical field of biology, and particularly discloses a bacillus cereusThe detection kit is used for real-time fluorescent RPA detection of Bacillus cereus and comprises a pair of primers and a probe based on a Bacillus cereus 16sRNA gene sequence. The detection method has excellent specificity and sensitivity, and the detection limit is 1.0 multiplied by 10‑3ng/μ L, when the pollution amount is more than or equal to 1.5 multiplied by 104And (3) detecting the bacillus cereus by real-time fluorescence RPA at CFU/g, wherein the required time is only 6-13 min. Therefore, the real-time fluorescent RPA established by the invention can quickly, efficiently and sensitively detect the bacillus cereus, and provides a referential technical means for the detection of the bacillus cereus in public health events such as food processing quality control and food-borne epidemic diseases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection kit for bacillus cereus, a detection method and application.
Background
Bacillus cereus (Bacillus cereus) is one of the main causes of food poisoning as an opportunistic pathogen, and can cause clinical symptoms mainly characterized by nausea, vomiting, abdominal pain and diarrhea. The bacteria are widely distributed in soil, sewage and air, and are also common in various raw or cooked animal and plant foods. The bacillus cereus has the characteristic of diversified metabolic pathways, has ecological adaptability and strong viability in different environments, can resist adverse factors, and can be better spread. The food is easy to pollute the bacillus cereus due to non-standard processing or improper storage, and after the polluted bacillus cereus or residual spores grow and reproduce, the polluted food is eaten by mistake because the appearance and the properties of the polluted food are not obviously changed, thereby causing food poisoning. The incidence of bacillus cereus poisoning is high and can reach 60-100%. Therefore, establishing a simple and rapid method for detecting bacillus cereus becomes a hotspot for researching the detection method of the bacillus cereus.
Recombinase Polymerase Amplification (RPA) is a nucleic acid Amplification technology first reported in 2006, and has the characteristics of simple operation and rapid reaction, so that the RPA is widely concerned in medical diagnosis, food-borne pathogenic bacteria, transgenic food and animal epidemic disease detection. The RPA technology mainly depends on recombinase, single-chain binding protein and DNA polymerase with strand displacement activity to be carried out under a constant temperature condition (25-42 ℃), and the real-time RPA technology combines the RPA basic reaction with a fluorescent probe, has the advantage of monitoring the amplification process in real time, further improves the sensitivity and specificity, and is widely applied to the detection of bacteria, viruses and parasites.
The currently reported detection methods of bacillus cereus comprise a separation culture method, a common PCR, a multiplex PCR, a fluorescence PCR, LAMP, a gene chip and the like, but the separation culture method and the gene chip technology are complex to operate, and the detection time is relatively long; the PCR technique is not only environmentally demanding, but also requires expensive PCR instruments and skilled technicians. The method has a lot of inconvenience in application in public health incidents such as sudden food poisoning. The LAMP method belongs to an isothermal detection method, but the LAMP reaction temperature is higher than 60 ℃, and the reaction time is longer, generally between 30-60 min.
Disclosure of Invention
Aiming at the problems of complicated operation, long detection time, high cost and the like of the existing bacillus cereus detection method, the invention provides a detection kit for bacillus cereus.
Further, the invention also provides a detection method for the bacillus cereus and application thereof.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the inventors of the present invention analyzed the 16sRNA gene sequence (accession number: KY224970.1) of Bacillus cereus in GeneBank, selected the conserved region, and designed real-time fluorescent RPA, i.e., real-time RPA primer and exo probe, so that the target fragment size was 297 bp.
A detection kit for detecting Bacillus cereus is used for real-time fluorescent RPA detection of the Bacillus cereus and comprises a pair of primers and a probe based on a Bacillus cereus 16sRNA gene sequence,
wherein, the sequences of the pair of primers and the probe are as follows:
an upstream primer: 5'-ATACCCTGGTAGTCCACGCCGTAAACGATGAG-3'
A downstream primer: 5'-CAACATCTCACGACACGAGCTGACGACAACCA-3'
And (3) probe: 5' -CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGC-FAM-dT-THF-
AAG-BHQ1-dT-TAACGCATTAAGC-C3-3'。
Further, the invention also provides a detection method for the bacillus cereus, wherein the detection method is a real-time fluorescence RPA method, and the detection method at least comprises the following steps:
and 3, determining whether the bacillus cereus exists in the sample to be detected or not by judging whether the reaction result is positive or not.
Furthermore, the invention also provides application of the detection kit or the detection method in the detection field of bacillus cereus.
Compared with real-time PCR, the real-time RPA method established by the invention has the advantages of simple and convenient operation and short reaction time, and meanwhile, an OptiGene III isothermal amplification fluorescent detection system used in the research is compact, small, light and durable, is only about 1.75kg, is operated by a touch screen, does not need to be connected with a computer, is internally provided with a long-endurance lithium battery, can work outdoors all day long in a powerless environment, and can realize portable pathogenic bacteria field rapid detection. Compared with LAMP method, although LAMP and real-time RPA both belong to isothermal detection method, LAMP reaction temperature is higher and higher than 60 ℃, reaction time is longer, generally between 30-60min, real-time RPA amplification can be realized at normal temperature (25-42 ℃), specificity is good, and detection sensitivity to genome DNA can be realizedUp to 1.0 × 10-3ng/mu L, simple operation, can be finished within 20min, and provides a valuable reference method for the technical field of rapid detection of bacillus cereus.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graphical representation of the results of real-time RPA sensitivity tests provided by embodiments of the present invention;
FIG. 2 is a graphical representation of the results of real-time PCR sensitivity tests provided by comparative examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A detection kit for detecting Bacillus cereus is used for real-time fluorescent RPA detection of the Bacillus cereus and comprises a pair of primers and a probe based on a Bacillus cereus 16sRNA gene sequence,
wherein, the sequences of the pair of primers and the probe are as follows:
an upstream primer: 5'-ATACCCTGGTAGTCCACGCCGTAAACGATGAG-3'
A downstream primer: 5'-CAACATCTCACGACACGAGCTGACGACAACCA-3'
And (3) probe: 5' -CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGC-FAM-dT-THF-
AAG-BHQ1-dT-TAACGCATTAAGC-C3-3'。
Preferably, the detection kit further comprises a lyophilized enzyme preparation and a magnesium acetate solution.
Preferably, the reaction system of the detection kit consists ofThe following were used: mu.L of 10. mu.M forward primer, 2. mu.L of 10. mu.M reverse primer, 0.6. mu.L of 10. mu.M probe, 12.5. mu.L of 20% polyethylene glycol, 1. mu.L of viral DNA template, 29.4. mu.L of ddH2O。
Preferably, the lyophilized enzyme preparation comprises 1mM deoxyribonucleoside triphosphate, 90 ng/. mu.L single-stranded binding protein, 120 ng/. mu.L recA recombinase, 30 ng/. mu.L Bsu DNA polymerase, 30 ng/. mu.L Exo-exonuclease, 100mmol/L trihydroxymethyl glycine, polyethylene glycol at a concentration of 20% by volume, 5mM dithiothreitol, and 100 ng/. mu.L creatine kinase.
The invention also provides a detection method for the bacillus cereus, which is a real-time fluorescence RPA method and at least comprises the following steps:
and 3, determining whether the bacillus cereus exists in the sample to be detected or not by judging whether the reaction result is positive or not.
Preferably, the amplification reaction is performed in an isothermal amplification fluorescence detector set at a temperature of 39 ℃ for 20 min.
Preferably, the final concentration of the upstream primer and the final concentration of the downstream primer of the bacillus cereus are both 0.4 mu M, and the final concentration of the probe is 0.12 mu M.
Preferably, the extraction of the DNA of bacillus cereus comprises the following steps: inoculating Bacillus cereus pure culture in 8-12mL nutrient broth, and culturing at 35-37 deg.C for 14-17 h; then 1mL of bacterial liquid is taken and centrifuged for 1min at 10000rpm, and the supernatant is discarded; and (3) extracting bacterial genome DNA from the bacterial precipitation according to a bacterial DNA extraction kit method, and immediately using the bacterial genome DNA after extraction or storing the bacterial genome DNA at the temperature of minus 20 ℃ for later use.
Preferably, the nucleic acid concentration of the extracted bacterial genomic DNA is determined using a ultramicro spectrophotometer.
The invention also provides application of the detection kit or the detection method in the field of bacillus cereus detection.
The following examples are provided to better illustrate the embodiments of the present invention.
This example provides a detection kit and a detection method for bacillus cereus, and the main reagents and devices used are: mannitol vitellocolin agar (MYP), nutrient broth, available from Beijing Luqiao technology, LLC; a bacterial genome DNA extraction kit purchased from Tiangen Biochemical technology (Beijing) Ltd; premix Ex Taq, available from Bao bioengineering (Dalian) Inc.; the RAA kit (fluorescent type) was purchased from Zhejiang Taijing Biotech limited; the primers and probes were synthesized by Biotechnology engineering (Shanghai) Inc.
Genie III isothermal amplification fluorescent detection System, OptiGene, UK; ABI7500 real-time fluorescent PCR instrument, ABI corporation, usa; NanoDrop 2000C ultramicro spectrophotometer, Thermo Scientific, USA.
Example 1 detection method of Bacillus cereus-establishment of real-time fluorescence RPA method
1. Design and preparation of primer and probe sequence
The inventors of the present invention analyzed the 16sRNA gene sequence (accession number: KY224970.1) of Bacillus cereus in GeneBank, selected the conserved region, and designed real-time fluorescent RPA, i.e., real-time RPA primer and exo probe, so that the target fragment size was 297 bp.
The present invention designs and synthesizes a pair of primers and a probe, as shown in Table 1.
TABLE 1 Bacillus cereus real-time RPA primers and probes designed by the present invention
2. Bacillus cereus strain and extraction of bacterial genome DNA
The bacillus cereus used in this study was kept in the laboratory.
Inoculating pure culture strain of Bacillus cereus (CICC63301) into 10mL of nutrient broth, and culturing at 37 deg.C for 16 h; centrifuging 1mL of bacterial liquid at 10000rpm for 1min, and removing supernatant; extracting bacterial genome DNA from the thallus precipitate according to a bacterial DNA extraction kit method, immediately using or storing at-20 ℃ for later use, and simultaneously measuring the nucleic acid concentration of the extracted Bacillus cereus DNA by using a NanoDrop 2000C ultramicro spectrophotometer.
3. Preparation of bacillus cereus detection kit
A single reaction system was prepared using the RAA kit (fluorescent type) as follows: mu.L of 10. mu.M forward primer (final concentration of 0.4. mu.M), 2. mu.L of 10. mu.M reverse primer (final concentration of 0.4. mu.M), 0.6. mu.L of 10. mu.M probe (final concentration of 0.12. mu.M), 12.5. mu.L of 20% by volume polyethylene glycol, 1. mu.L of viral DNA template, 29.4. mu.L of ddH2And O. And uniformly mixing 47.5 mu L of the system, adding the mixed system into a reaction tube filled with the freeze-dried enzyme preparation, blowing and beating the mixed system up and down by using a pipette until the mixed system is completely dissolved, adding 2.5 mu L of 280mM magnesium acetate into the reaction tube, performing instantaneous centrifugation and vortex, and putting the mixture into an isothermal amplification fluorescence detector Genie III, wherein the temperature is set to be 39 ℃, and the reaction time is 20 min.
The freeze-dried enzyme preparation comprises 1mM of deoxyribonucleoside triphosphate, 90 ng/. mu.L of single-chain binding protein, 120 ng/. mu.L of recA recombinase, 30 ng/. mu.L of Bsu DNA polymerase, 30 ng/. mu.L of Exo exonuclease, 100mmol/L of trihydroxymethyl glycine, polyethylene glycol with the volume concentration of 20%, 5mM of dithiothreitol and 100 ng/. mu.L of creatine kinase.
4. Establishment of real-time RPA method as bacillus cereus detection method
The establishment of the detection method comprises the following steps:
the amplification reaction is carried out in an isothermal amplification fluorescence detector with the temperature set to 37-39 ℃, and the reaction time is 20-30 min;
and 4, determining whether the bacillus cereus exists in the sample to be detected by judging whether the reaction result is positive.
To better illustrate the establishment of the real-time fluorescent RPA method for Bacillus cereus detection provided in the examples of the present invention, the real-time RPA method established in example 1 was specifically tested.
In order to better illustrate the technical solution of the present invention, further comparison is made below by means of a comparative example and an example of the present invention.
Comparative example 1 establishment of real-time PCR method for Bacillus cereus detection method
1. A pair of primers and probes for real-time PCR are shown in Table 2.
TABLE 2 Bacillus cereus real-time PCR primers and probes designed by the present invention
2. Establishment of real-time PCR method
The reaction system is as follows: 1.5. mu.L of 10. mu.M forward primer (final concentration of 0.6. mu.M), 2. mu.L of 10. mu.M reverse primer (final concentration of 0.6. mu.M), 1. mu.L of 5. mu.M probe (final concentration of 0.2. mu.M), 12.5. mu.L of Premix Ex Taq, 1. mu.L of viral DNA template, and 7.5. mu.L of ddH2O。
The system is fully and uniformly mixed and then put into an ABI7500 real-time fluorescent PCR instrument, and the reaction program is set as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, extension at 60 ℃ for 35s, 35 cycles, and fluorescence signal collection at 60 ℃.
To better illustrate the application of the real-time fluorescent RPA method for Bacillus cereus detection provided in the examples of the present invention, example 1 and comparative example 1 were conducted as specificity and sensitivity tests and tests for detection of artificially contaminated samples, respectively.
Specificity test
The real-time RPA reaction was performed using genomic DNA of the strains shown in Table 3 as a template to determine whether a specific amplification curve was present.
TABLE 3
Note: +, positive result; negative results.
As can be seen from the results in Table 3, real-time RPA detection is carried out on the genomic DNA of the Bacillus cereus and other bacteria as templates, and the results show that only the Bacillus cereus presents a typical amplification curve and is a positive result, while no amplification curve appears in other Bacillus and non-Bacillus bacteria, and the results show that the method has good specificity.
Sensitivity test
Bacillus cereus genomic DNA at a concentration of 100 ng/. mu.L was subjected to ddH2O10-fold gradient dilution, 8 gradients serially diluted, and real-time RPA and real-time PCR detection using different dilutions of DNA as templates, the results are shown in FIG. 1 and FIG. 2.
FIG. 1 shows the real-time RPA detection result, and FIG. 2 shows the real-time PCR detection result. 1: 1.0X 10 in FIGS. 1 and 22ng/μL；2:1.0×101ng/μL；3:1.0×100ng/μL；4:1.0×10-1ng/μL；5:1.0×10-2ng/μL；6:1.0×10-3ng/μL；7:1.0×10-4ng/μL；8:1.0×10-5ng/μL。
The detection results of FIGS. 1 and 2 show that the detection limit of real-time RPA is 1.0X 10-3ng/uL, which is consistent with the detection limit of the real-time PCR method.
Test for detection of artificially contaminated sample
Carrying out overnight pure culture on Bacillus cereus (CICC63301), and carrying out 10-fold gradient dilution with physiological saline, selecting 10-5、10-6、10-7Three dilutions of 200. mu.L of inoculum were spread on MYP agar plates and 3 replicates were taken for calculation of the initial concentration of pure cultures. Adding 1mL of bacterial liquids with different dilutions into 25g of rice samples (the samples are detected according to GB4789.14-2014 in advance, bacillus cereus is not detected), adding the samples into 225mL of PBS, continuously homogenizing for 1-2min by using a tapping homogenizer, extracting bacterial DNA by taking 1mL of bacterial liquids from the samples according to the extraction method of the bacterial genome DNA provided by the invention, taking 1 muL of bacterial liquids as a template to perform real-time RPA and real-time PCR detection, wherein the detection results are shown in Table 4.
TABLE 4 test results of the detection of artificially contaminated samples
Note: -, no detection.
As can be seen from Table 4, when the amount of contamination was 1.5X 10 or more4When CFU/g is adopted, real-time RPA can detect the bacillus cereus in the rice, and the required time is only 6-13 min; when the pollution amount is more than or equal to 1.5 multiplied by 105The CFU/g, real-time PCR method can detect the bacillus cereus in the rice, and the required time is more than 30min (Ct value is 20-31). The results show that the detection time of real-time RPA is obviously lower than that of real-time PCR when detecting artificially contaminated samples, and the sensitivity of real-time RPA is higher than that of real-time PCR.
Food processed products such as rice are the most important food species for bacillus cereus food poisoning. The real-time RPA method established in the invention can effectively detect the polluted bacillus cereus in the rice, the detection sensitivity is superior to that of real-time PCR, and the reaction time is obviously shorter than that of the real-time PCR. The successful establishment of the method combines the rapid extraction of bacterial nucleic acid, can achieve the aim of on-site rapid detection, and provides a reference technical means for the detection of the bacillus cereus in public health events such as food processing quality control and food-borne epidemic diseases.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
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Claims (4)
1. A detection kit for Bacillus cereus is characterized in that: the detection kit is used for real-time fluorescent RPA detection of Bacillus cereus, and comprises a pair of primers and a probe based on a Bacillus cereus 16sRNA gene sequence,
wherein, the sequences of the pair of primers and the probe are as follows:
an upstream primer: 5'-ATACCCTGGTAGTCCACGCCGTAAACGATGAG-3'
A downstream primer: 5'-CAACATCTCACGACACGAGCTGACGACAACCA-3'
And (3) probe: 5' -CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGC-FAM-dT-THF-
AAG-BHQ1-dT-TAACGCATTAAGC-C3-3'。
2. The detection kit for bacillus cereus according to claim 1, wherein: the detection kit also comprises a freeze-dried enzyme preparation and a magnesium acetate solution.
3. The detection kit for bacillus cereus according to claim 1 or 2, wherein: the reaction system of the detection kit comprises the following components: 2 μ L of 10 μM upstream primer, 2. mu.L of 10. mu.M downstream primer, 0.6. mu.L of 10. mu.M probe, 12.5. mu.L of 20% polyethylene glycol by volume, 1. mu.L of viral DNA template, 29.4. mu.L of ddH2O。
4. The detection kit for bacillus cereus according to claim 2, wherein: the freeze-dried enzyme preparation comprises 1mM of deoxyribonucleoside triphosphate, 90 ng/. mu.L of single-chain binding protein, 120 ng/. mu.L of recA recombinase, 30 ng/. mu.L of Bsu DNA polymerase, 30 ng/. mu.L of Exo exonuclease, 100mmol/L of trihydroxymethyl glycine, polyethylene glycol with the volume concentration of 20%, 5mM of dithiothreitol and 100 ng/. mu.L of creatine kinase.
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| CN103667251A (en) * | 2012-09-03 | 2014-03-26 | 中国科学院上海生命科学研究院 | Method for detecting food-borne pathogenic bacteria at high throughput |
| CN106636459A (en) * | 2016-10-18 | 2017-05-10 | 河北出入境检验检疫局检验检疫技术中心 | Fluorescent RT-RPA specific detection of American porcine reproductive and respiratory syndrome virus |
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| CN103667251A (en) * | 2012-09-03 | 2014-03-26 | 中国科学院上海生命科学研究院 | Method for detecting food-borne pathogenic bacteria at high throughput |
| CN106636459A (en) * | 2016-10-18 | 2017-05-10 | 河北出入境检验检疫局检验检疫技术中心 | Fluorescent RT-RPA specific detection of American porcine reproductive and respiratory syndrome virus |
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