CN107236812A - Detection kit, detection method and application for bacillus cereus - Google Patents
Detection kit, detection method and application for bacillus cereus Download PDFInfo
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Abstract
The present invention relates to biological technical field, a kind of detection kit, detection method and application for bacillus cereus is specifically disclosed, the detection kit is used for real-time fluorescence RPA and detects bacillus cereus, including the pair of primers based on bacillus cereus 16sRNA gene orders and a probe.The detection method has excellent specificity and sensitivity, and detection is limited to 1.0 × 10‑3Ng/ μ L, when contaminant capacity >=1.5 × 104During CFU/g, real-time fluorescence RPA can detect bacillus cereus, and the required time is only 6 13 min.Therefore the real-time fluorescence RPA energy of the invention set up is quick, efficient, sensitively detect bacillus cereus, and the detection for bacillus cereus in the public health event such as food processing quality control and food-borne epidemic disease provides referential technological means.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of detection kit for bacillus cereus, detection
Method and application.
Background technology
Bacillus cereus (Bacillus cereus), as a kind of opportunistic pathogenic bacteria, is the main original of food poisoning
Therefore one, it can cause with Nausea and vomiting, stomachache and suffer from diarrhoea as the clinical symptoms of principal character.The bacterium be distributed widely in soil,
It is also common in all kinds of lifes or ripe animals and plants food in dirty water and air.Bacillus cereus has metabolic pathway variation
The characteristics of, possess ecological adaptability and powerful survival ability in different environment, unfavorable factor can be resisted, preferably
Propagate.Due to processing, lack of standardization or storage is improper easily pollutes bacillus cereus, the bacillus cereus of pollution or residual to food
After the gemma growth and breeding deposited, because the food appearance and character of pollution are eaten by mistake without significant change, so as to cause food poisoning.
The incidence of disease of bacillus cereus poisoning is higher, up to 60-100%.Therefore, set up simplicity, quickly detect waxy gemma bar
The method of bacterium turns into the focus for studying the bacterium detection method.
Recombinase polymeric enzymatic amplification technology (Recombinase Polymerase Amplification, RPA) is 2006
Year the nucleic acid amplification technologies reported first, because its is simple to operate, the characteristics of react quick, medical diagnosis, food-borne pathogens,
Received significant attention in GM food and animal epidemic detection.RPA technologies depend on recombinase, single strand binding protein and
(25-42 DEG C) progress under constant temperature of archaeal dna polymerase with strand-displacement activity, and real-time RPA technologies are by RPA bases
Plinth reaction is combined with fluorescence probe, and the advantage for possessing real-time monitoring amplification process, sensitivity and specificity are also further improved,
It has been widely used in the detection of bacterium, virus and parasite.
At present the bacillus cereus detection method reported have isolated culture, regular-PCR, multiplex PCR, fluorescent PCR,
LAMP and genetic chip etc., but isolated culture and biochip technology are cumbersome, detection time is relatively long；PCR skills
Art is not only high to environmental requirement, and needs PCR instrument device and the those skilled in the art of costliness.The above method is in burst food
Application in the public health events such as poison there are inconvenience.LAMP method belongs to isothermal detection methods, but LAMP reaction temperature
Degree is higher, and higher than 60 DEG C, and the reaction time is longer, typically between 30-60min.
The content of the invention
For existing bacillus cereus detection method is cumbersome, detection time is long, the problems such as cost is high, the present invention is carried
For a kind of detection kit for bacillus cereus.
Further, the present invention also provides detection method and its application for bacillus cereus.
To achieve the above object of the invention, the embodiment of the present invention employs following technical scheme：
Inventor passes through the 16sRNA gene order (accession number for bacillus cereus in GeneBank：
KY224970.1) analyzed, select conservative region, design real-time fluorescence RPA is real-time RPA primers and exo probes,
Purpose fragment size is 297bp.
A kind of detection kit of bacillus cereus, the detection kit is used for real-time fluorescence RPA and detects waxy bud
Spore bacillus, including the pair of primers based on bacillus cereus 16sRNA gene orders and a probe,
Wherein, the sequence of described pair of primers and a probe is as follows：
Sense primer：5'-ATACCCTGGTAGTCCACGCCGTAAACGATGAG-3'
Anti-sense primer：5'-CAACATCTCACGACACGAGCTGACGACAACCA-3'
Probe：5'-CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGC-FAM-dT-THF-
AAG-BHQ1-dT-TAACGCATTAAGC-C3-3'。
Further, the present invention also provides a kind of detection method for bacillus cereus, and the detection method is real
When fluorescence RPA methods, the detection method at least comprises the following steps：
Step 1, genomic DNA is extracted from testing sample；
Step 2, the DNA extracted using the above-mentioned detection kit for bacillus cereus to step 1 are carried out
Isothermal amplification；Wherein, the temperature of the isothermal amplification is 37-39 DEG C, and the time is 20-30min；
Step 3, by judging whether reaction result is positive, determine to whether there is bacillus cereus in testing sample.
Further, the present invention also provides described detection kit or detection method is detected in bacillus cereus
Application in field.
The real-time RPA methods that the present invention is set up compared with real-time PCR, with it is easy to operate, reaction when
Between short advantage, it is light resistance to while the OptiGene III isothermal duplication fluorescence detecting systems used in this research are compact compact
With, only 1.75kg or so, touch screen operation, without connecting computer, its built-in long endurance lithium battery can be in non-transformer environment
Whole day outwork, can be achieved portable pathogenic bacteria field quick detection.Compared with LAMP method, although LAMP and real-
TimeRPA belongs to isothermal detection methods, but LAMP reaction temperature higher reaction temperatures are higher, higher than 60 DEG C, and during reaction
Between it is longer, typically between 30-60min, and (25-42 DEG C) is that can be achieved at normal temperatures for real-time RPA amplifications, and is had
Good specificity, to the detection sensitivity of genomic DNA up to 1.0 × 10-3Ng/ μ L, it is simple to operate, in 20min
Complete, valuable reference method is provided for bacillus cereus rapid detection technical field.
Brief description of the drawings
Technical scheme in order to illustrate the embodiments of the present invention more clearly, below by using required in embodiment
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for ability
For the those of ordinary skill of domain, on the premise of not paying creative work, it can also be obtained according to these accompanying drawings other attached
Figure.
Fig. 1 is real-time RPA sensitivitys result of the test schematic diagram provided in an embodiment of the present invention；
Fig. 2 is the real-time PCR sensitivity result of the test schematic diagrames that comparative example of the present invention is provided.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
A kind of detection kit for bacillus cereus, the detection kit is used for real-time fluorescence RPA and detects wax
Sample bacillus, including the pair of primers based on bacillus cereus 16sRNA gene orders and a probe,
Wherein, the sequence of described pair of primers and a probe is as follows：
Sense primer：5'-ATACCCTGGTAGTCCACGCCGTAAACGATGAG-3'
Anti-sense primer：5'-CAACATCTCACGACACGAGCTGACGACAACCA-3'
Probe：5'-CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGC-FAM-dT-THF-
AAG-BHQ1-dT-TAACGCATTAAGC-C3-3'。
Preferably, the detection kit also includes lyophilized enzyme preparation and magnesium acetate solution.
Preferably, the reaction system composition of the detection kit is as follows：2 μ L 10 μM of sense primer, 10 μM of 2 μ L
Anti-sense primer, 0.6 μ L 10 μM of probe, 12.5 μ L volumetric concentration is 20% polyethylene glycol, 1 μ L viral DNA mould
Plate, 29.4 μ L ddH2O。
Preferably, the lyophilized enzyme preparation includes 1mM deoxyribonucleoside triphosphate, 90ng/ μ L single-stranded combination egg
In vain, 120ng/ μ L recA recombinases, 30ng/ μ L Bsu archaeal dna polymerases, 30ng/ μ L Exo exonucleases,
100mmol/L trihydroxy methyl glycine, volumetric concentration be 20% polyethylene glycol, 5mM dithiothreitol (DTT), 100ng/ μ L's
Creatine kinase.
The present invention also provides a kind of detection method for bacillus cereus, and the detection method is real-time fluorescence RPA
Method, the detection method at least comprises the following steps：
Step 1, genomic DNA is extracted from testing sample；
Step 2, the DNA extracted using the above-mentioned detection kit for bacillus cereus to step 1 are carried out
Isothermal amplification；Wherein, the temperature of the isothermal amplification is 37-39 DEG C, and the time is 20-30min；
Step 3, by judging whether reaction result is positive, determine to whether there is bacillus cereus in testing sample.
Preferably, amplified reaction is carried out in the isothermal duplication fluorescence detector that temperature is set as 39 DEG C, and the reaction time is
20min。
Preferably, the final concentration of the upstream and downstream primer of the bacillus cereus is 0.4 μM, probe it is final concentration of
0.12μM。
Preferably, the DNA of bacillus cereus extraction comprises the following steps：Will be by bacillus cereus pure culture
Bacterium is inoculated in 8-12mL nutrient broths, and 14-17h is cultivated at 35-37 DEG C；Then take 1mL bacterium solutions with 10000rpm rotating speeds from
Heart 1min, abandons supernatant；Bacterial sediment is extracted into bacterial genomes DNA according to DNA of bacteria extracts reagent cassette method, extraction is finished
Afterwards immediately using or -20 DEG C save backup.
Preferably, extracted bacterial genomes DNA nucleic acid concentration is determined using ultramicrospectrophotometer.
The present invention also provides described detection kit or detection method in bacillus cereus detection detection field
Using.
It is provided in an embodiment of the present invention in order to better illustrate, illustrated below by embodiment is further.
The present embodiment provides the detection kit and detection method for bacillus cereus, main agents used with
Equipment has：Mannitol yolk polymyxins agar (MYP), nutrient broth, purchased from Beijing Luqiao Technology Co., Ltd.；Bacterium
Genome DNA extracting reagent kit, purchased from TIANGEN Biotech (Beijing) Co., Ltd.；Premix Ex Taq, purchased from precious biological work
Journey (Dalian) Co., Ltd；RAA kits (fluorescent type) are purchased from Zhejiang Taijing Biotechnology Co., Ltd.；Primer and probe are by giving birth to
Work bioengineering (Shanghai) limited company synthesizes.
Genie III isothermal duplication fluorescence detecting systems, OptiGene companies of Britain；ABI7500 real-time fluorescence PCR instrument,
American AB I companies；NanoDrop 2000C ultramicrospectrophotometers, Thermo Scientific companies of the U.S..
The foundation of the detection method of the bacillus cereus of embodiment 1-real-time fluorescence RPA methods
1st, the design and preparation of primer and probe sequence
Inventor passes through the 16sRNA gene order (accession number for bacillus cereus in GeneBank：
KY224970.1) analyzed, select conservative region, design real-time fluorescence RPA is real-time RPA primers and exo probes,
Purpose fragment size is 297bp.
Present invention design has synthesized pair of primers and a probe, as shown in table 1.
The bacillus cereus real-time RPA primer and probes that the present invention of table 1 is designed
2nd, Bacillus cereus strain and bacterial genomes DNA extraction
The bacillus cereus that this research is used preserves by this laboratory.
Bacillus cereus (CICC63301) pure culture bacterium is inoculated in 10mL nutrient broths, cultivated at 37 DEG C
16h；Take 1mL bacterium solutions to centrifuge 1min with 10000rpm rotating speed, abandon supernatant；By bacterial sediment according to DNA of bacteria extracts reagent
Cassette method extract bacterial genomes DNA, immediately using or -20 DEG C save backup, while using NanoDrop 2000C ultramicron
The nucleic acid concentration for the bacillus cereus DNA that spectrophotometric determination is extracted.
3rd, the preparation of bacillus cereus detection kit
Prepare as follows in single reaction system, reaction system using RAA kits (fluorescent type)：2 μ L 10 μM of upstream
Primer (final concentration of 0.4 μM), 2 μ L 10 μM of anti-sense primer (final concentration of 0.4 μM), 0.6 μ L 10 μM of probe is (dense eventually
Spend for 0.12 μM), 12.5 μ L volumetric concentration is 20% polyethylene glycol, 1 μ L viral DNA template, 29.4 μ L ddH2O。
It is added to after will above-mentioned 47.5 μ L systems be mixed in the reaction tube equipped with lyophilized enzyme preparation, with being blown and beaten above and below pipettor to complete
Dissolving, isothermal duplication fluorescence is put into after 2.5 μ L 280mM magnesium acetate, brief centrifugation and vortex are then added into reaction tube
In detector Genie III, temperature is set as 39 DEG C, and the reaction time is 20min.
The lyophilized enzyme preparation includes 1mM deoxyribonucleoside triphosphate, 90ng/ μ L single strand binding protein,
120ng/ μ L recA recombinases, 30ng/ μ L Bsu archaeal dna polymerases, 30ng/ μ L Exo exonucleases, 100mmol/L
Trihydroxy methyl glycine, volumetric concentration be 20% polyethylene glycol, 5mM dithiothreitol (DTT), 100ng/ μ L creatine kinase.
4th, the foundation of bacillus cereus detection method-real-time RPA methods
The foundation of the detection method comprises the following steps：
Step 1, genomic DNA is extracted from testing sample；
Step 2, the DNA extracted to step 1 carry out isothermal amplification；Wherein reaction solution is taken above-mentioned waxy
The upstream and downstream primer and probe of bacillus, the reaction solution is mixed, and is added in the reaction tube equipped with lyophilized enzyme preparation,
Dissolving, then 2.5 μ L 280mM magnesium acetate solution is added into reaction tube, carry out amplified reaction；
Amplified reaction is carried out in the isothermal duplication fluorescence detector that temperature is set as 37-39 DEG C, and the reaction time is 20-
30min；
Step 4, by judging whether reaction result is positive, determine to whether there is bacillus cereus in testing sample.
In order to better illustrate the real-time fluorescence RPA methods provided in an embodiment of the present invention detected for bacillus cereus
Foundation, below by the real-time RPA methods that embodiment 1 is set up do specificity experiment.
In order to better illustrate technical scheme, done further below by comparative example and embodiments of the invention into one
The contrast of step.
Comparative example 1 is used for the foundation of bacillus cereus detection method-real-time PCR methods
1st, real-time PCR pair of primers and probe as shown in table 2
Bacillus cereus real-time PCR primers and probe that the present invention of table 2 is designed
2nd, the foundation of real-time PCR methods
Reaction system is：1.5 μ L 10 μM of sense primer (final concentration of 0.6 μM), 2 μ L 10 μM of anti-sense primer
(final concentration of 0.6 μM), 1 μ L 5 μM of probe (final concentration of 0.2 μM), 12.5 μ L Premix Ex Taq, 1 μ L virus
DNA profiling, 7.5 μ L ddH2O。
It is put into after above-mentioned system is fully mixed in ABI7500 real-time fluorescence PCR instrument, response procedures are set to：95 DEG C, in advance
It is denatured 30s；95 DEG C of denaturation 5s, 60 DEG C of extension 35s, 35 circulations, 60 DEG C of collection fluorescence signals.
In order to better illustrate the real-time fluorescence RPA methods provided in an embodiment of the present invention detected for bacillus cereus
Application, below by embodiment 1 and comparative example 1 makees respectively specificity, sensitivity test and artificial contamination's sample detection examination
Test.
Specific test
Real-time RPA reactions are carried out using the genomic DNA of bacterial strain shown in table 3 as template, it is determined whether occur
Specific amplification curve.
Table 3
Note：+, positive findings；-, negative findings.
As seen from the results in Table 3, real-time is carried out as template to bacillus cereus and other bacterial genomes DNA
RPA is detected, is as a result shown that only typical amplification curve is presented in bacillus cereus, is positive findings, and other bacillus and
Non- bacillus bacterium does not occur amplification curve, as a result illustrates that this method has good specificity.
Sensitivity is tested
Concentration is used into ddH for 100ng/ μ L bacillus cereus genomic DNA2O carries out 10 times of gradient dilutions, continuously
Dilute after 8 gradients, real-time RPA and real-time PCR detections carried out using the DNA of different dilution factors as template,
As a result as shown in 1 and Fig. 2.
Fig. 1 is real-time RPA testing results, and Fig. 2 is real-time PCR testing results.1 in Fig. 1,2:1.0×
102ng/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。
Show that real-time RPA detection is limited to 1.0 × 10 from Fig. 1,2 testing result-3Ng/ μ L, same to real-
Time PCR method test limits are consistent.
The experiment of artificial contamination's sample detection
By Bacillus cereus (CICC63301) after pure culture overnight, 10 times of gradient dilutions are carried out with physiological saline, are selected
Take 10-5、10-6、10-7The μ L of bacterium solution 200 of three dilution factors are applied on MYP agar plates, and make 3 parallel samples, for calculating
The initial concentration of pure culture bacterium.The bacterium solution 1mL of different dilution factors is respectively added in 25g rice samples to (sample is pressed in advance
According to GB4789.14-2014 detections, Bacillus cereus is not detected), it is then added in 225mL PBS, is connected with slap type homogenizer
Continuous homogeneous 1-2min, therefrom respectively takes 1mL bacterium solutions to carry out bacterium with reference to the extracting method of the bacterial genomes DNA of the invention provided
DNA extraction, takes 1 μ L to carry out real-time RPA and real-time PCR as template and detects, the testing result such as institute of table 4
Show.
The testing inspection result of the artificial contamination's sample detection of table 4
Note：-, do not detect.
As can be seen from Table 4, when contaminant capacity >=1.5 × 104During CFU/g, real-time RPA can be detected in rice
Bacillus cereus, the required time is only 6-13min；When contaminant capacity >=1.5 × 105CFU/g, real-time PCR side can
Detect bacillus cereus in rice, required time is more than 30min (Ct values are 20-31).As a result illustrate, it is artificial in detection
Real-time RPA detection time is significantly lower than real-time PCR detection time, real-time during contaminated samples
RPA sensitivity is higher than real-time PCR.
The grain processing product such as rice are the topmost food varieties of bacillus cereus food poisoning.Set up in the present invention
Real-time RPA methods can effectively detect the bacillus cereus polluted in rice, and detection sensitivity is better than real-
Time PCR, the reaction time is also considerably less than real-time PCR.This method is successfully established, with reference to the quick of bacterial nucleic acid
Extract, the purpose of field quick detection can be reached, be the public health event such as food processing quality control and food-borne epidemic disease
The detection of middle bacillus cereus provides referential technological means.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modification, equivalent substitution or improvement made within refreshing and principle etc., should be included in the scope of the protection.
Sequence table
<110>Technology Center Of Hebei Import and Export Inspection and Quarantine Bureau
<120>Detection kit, detection method and application for bacillus cereus
<130> 2017
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Claims (10)
1. a kind of detection kit for bacillus cereus, it is characterised in that：The detection kit is used for real-time fluorescence
RPA detects bacillus cereus, including the pair of primers based on bacillus cereus 16sRNA gene orders and a probe,
Wherein, the sequence of described pair of primers and a probe is as follows：
Sense primer：5'-ATACCCTGGTAGTCCACGCCGTAAACGATGAG-3'
Anti-sense primer：5'-CAACATCTCACGACACGAGCTGACGACAACCA-3'
Probe：5'-CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGC-FAM-dT-THF-
AAG-BHQ1-dT-TAACGCATTAAGC-C3-3'。
2. it is used for the detection kit of bacillus cereus as claimed in claim 1, it is characterised in that：The detection kit
Also include lyophilized enzyme preparation and magnesium acetate solution.
3. it is used for the detection kit of bacillus cereus as claimed in claim 1 or 2, it is characterised in that：The detection examination
The reaction system composition of agent box is as follows：2 μ L 10 μM of sense primer, 2 μ L 10 μM of anti-sense primer, 10 μM of 0.6 μ L
Probe, 12.5 μ L volumetric concentration is 20% polyethylene glycol, 1 μ L viral DNA template, 29.4 μ L ddH2O。
4. it is used for the detection kit of bacillus cereus as claimed in claim 2, it is characterised in that：The lyophilized enzyme preparation
Deoxyribonucleoside triphosphate including 1mM, 90ng/ μ L single strand binding protein, 120ng/ μ L recA recombinases, 30ng/ μ
L Bsu archaeal dna polymerases, 30ng/ μ L Exo exonucleases, 100mmol/L trihydroxy methyl glycine, volumetric concentration is
20% polyethylene glycol, 5mM dithiothreitol (DTT), 100ng/ μ L creatine kinase.
5. a kind of detection method for bacillus cereus, it is characterised in that：The detection method is real-time fluorescence RPA side
Method, the detection method at least comprises the following steps：
Step 1, genomic DNA is extracted from testing sample；
Step 2, using being extracted described in claim any one of 1-4 for the detection kit of bacillus cereus to step 1
The DNA carry out isothermal amplification；Wherein, the temperature of the isothermal amplification is 37-39 DEG C, and the time is 20-
30min；
Step 3, by judging whether reaction result is positive, determine to whether there is bacillus cereus in testing sample.
6. it is used for the detection method of bacillus cereus as claimed in claim 5, it is characterised in that：The amplified reaction is in temperature
Degree is set as carrying out in 39 DEG C of isothermal duplication fluorescence detector, and the reaction time is 20min.
7. it is used for the detection method of bacillus cereus as claimed in claim 5, it is characterised in that：The isothermal amplification
Reaction system in, the final concentration of the upstream and downstream primer of bacillus cereus is 0.4 μM, final concentration of 0.12 μM of probe.
8. it is used for the detection method of bacillus cereus as claimed in claim 5, it is characterised in that：The bacillus cereus
DNA extraction comprise the following steps：Bacillus cereus pure culture bacterium will be inoculated in 8-12mL nutrient broths, in 35-
14-17h is cultivated at 37 DEG C；Then take 1mL bacterium solutions to centrifuge 1min with 10000rpm rotating speeds, abandon supernatant；By bacterial sediment according to
DNA of bacteria extracts reagent cassette method extract bacterial genomes DNA, extraction finish after immediately using or -20 DEG C save backup.
9. it is used for the detection method of bacillus cereus as claimed in claim 8, it is characterised in that：Use ultramicron light splitting light
Degree meter determines extracted bacterial genomes DNA nucleic acid concentration.
10. the detection kit or claim 5-9 for bacillus cereus as described in claim any one of 1-4 are any
Application of the detection method for bacillus cereus in bacillus cereus detects detection field described in.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111996266A (en) * | 2020-06-05 | 2020-11-27 | 江苏海洋大学 | Specific forward and reverse primers, probe and detection kit for vomit-causing bacillus cereus and application of specific forward and reverse primers and probe |
| CN112575097A (en) * | 2020-10-12 | 2021-03-30 | 南开大学 | Liquid phase chip for detecting bacillus cereus and application |
| CN112877448A (en) * | 2020-12-30 | 2021-06-01 | 广东省微生物研究所（广东省微生物分析检测中心） | Bacillus cereus standard strain containing specific molecular target and detection and application thereof |
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| 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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