CN102936621B - Bacillus cereus detection method and kit - Google Patents
Bacillus cereus detection method and kit Download PDFInfo
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- CN102936621B CN102936621B CN201210308753.XA CN201210308753A CN102936621B CN 102936621 B CN102936621 B CN 102936621B CN 201210308753 A CN201210308753 A CN 201210308753A CN 102936621 B CN102936621 B CN 102936621B
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Abstract
The present invention provides a new Bacillus cereus detection method, which comprises: 1) extracting DNA from a sample requiring detection; and 2) adopting the DNA obtained from the step 1) as a template to carry out real-time fluorescence quantitative PCR detection, wherein a target detected by the real-time fluorescence quantitative PCR detection is Bacillus cereus murB gene. The present invention further provides a Bacillus cereus detection kit, which comprises primers for Bacillus cereus murB gene amplification.
Description
Technical field
The invention belongs to the detection of nucleic acids in food safety field.Particularly, the present invention relates to method and the test kit of bacillus cereus rapid detection.
Background technology
Bacillus cereus (Bacillus cereus) is as the one in aerobic spore-bearing bacilli group, without pod membrane, can move, Gram-positive bacillus, gemma can tolerate 100 DEG C and reach 30 minutes.This bacterium is extremely wide in distributed in nature, is common in soil, sewage and dust; At 16-45 DEG C of energy growth and breeding, optimum growth temperature is 35 DEG C.Chang Yinben fungi pollution is rich in the food such as the starchbased product of carbohydrate and the milk-product of rich in proteins growth and breeding and cause food poisoning therein.Bacillus cereus is also the conditioned pathogen of the various non-infectious intestinal diseases of humans and animals, can cause that the mankind suffer from the diseases such as conjunctivitis, respiratory system, cardiovascular systems, central nervous system, wound infection.The main morbid substance of bacillus cereus is that two kinds of heat-resisting and heat-labile toxins that people is caused a disease, a large amount of viable bacteria and meta-bolites are as Phospholipid hydrolase, nuclease, hemolysin etc.Increase gradually as the case of the traumatic infection bacterium on skin bacillus cereus in recent years.Have now found that and also cause the similar gas gangrene of Burn Infection symptom, develop very soon, occur muscular death.Having whole body toxicity symptom, is by due to the extracellular toxin of this bacterium.This bacterial infection is invalid to any microbiotic.
At food enterprise, especially grain and oil processing enterprise, is faced with the Pollution abatement problem of serious bacillus cereus, but, putting a Tough questions before eyes is that the detection of bacillus cereus especially detection by quantitative is very difficult, makes the control of this biological hazard factor become empty talk.
Set up the quantitative detecting method of a kind of effective food source property bacillus cereus, to improve detection speed and the accuracy to this bacterium, contaminated food is processed in time, all significant in public health, food sanitation and port quarantine etc.
But traditional detection method is consuming time, complex operation, and there is based on the product of immunology principle the problems such as limit for height, specificity are not strong that detect.Especially bacillus cereus group's 6 kinds of bacterium, comprise bacillus cereus (B.cereus), Bacillus anthracis (B.anthracis), bacillus thuringiensis (B.thuringiensis), bacillus mycoides (B.mycoides), pseudomycete sample genus bacillus (B.pseudomycoides) and Webster genus bacillus (B.weihenstephanensis), phenotype and biochemical character similarity are very high, only producing enterotoxin kind, the aspect such as have or not of parasporal crystal there are differences, the existence of more not producing enterotoxin or not carrying the dissociant of parasporal crystal, make the discriminating of this group very difficult.
For this reason, the inventor is through studying for a long period of time, work out unexpectedly a kind of method of the lock nucleotide probe fluorescent quantitation rapid detection taking bacillus cereus murB as target, not only can effective monitoring pcr amplification, and the species specificity of bacillus cereus is strong, can effectively distinguish other genus bacillus of bacillus cereus group, fast, detection sensitivity is high, and can be applied to the high-throughput screening of bacillus cereus in food.
Summary of the invention
First aspect of the present invention is to provide the detection method of a kind of new bacillus cereus.This detection method comprises: 1) extract the DNA in detected sample; 2) using step 1) obtain DNA as template, carry out real-time fluorescence quantitative PCR detection; Wherein, the target that real-time fluorescence quantitative PCR detects is bacillus cereus murB gene.
Why the present invention screens murB gene to obtain by the following method as detecting target:
Download and collect by public data library searching, set up the gene pool about the genus bacillus of bacillus cereus and non-wax sample.Choose a bacillus cereus genome as target gene group, utilize program genomecut.pl to cut apart it, clip size is 100bp.Other bacillus cereus genomes in segmentation and kind are carried out to similarity analysis with BLAST, utilize program cofinder.pl to excavate conservative fragments (being the fragment that each bacillus cereus genome all contains) in kind.With BLAST, conservative fragments and outer other the non-bacillus cereus genomes of kind are carried out to similarity analysis again, utilize Programm pfinder.pl to excavate kind specific fragment (kind specific fragment judging criterion: in kind, conservative fragments and other all microbial genome homology regions of having checked order are no more than 25bp), thereby preliminary screening is to target sequence.
Use the method for genomics, first set up genomic library, then in object bacillus cereus genome, filtered out altogether totally 100 specific fragments by online BLAST, be i.e. 100 specific DNA sequences fragments that size is 100bp.These 100 specific fragments are organized into 87 through merging, design respectively the regular-PCR detection evaluation that primer carries out specificity and sensitivity, finally prove that primer specificity and sensitivity that murB gene pairs is answered are the best, therefore selected murB gene is for detecting target.
For several bacterium of bacillus cereus group are distinguished, the present invention is by the analysis in the conservative site of murB gene, the probe of distinctive murB gene conservative site being locked to Nucleotide (locked nucleic acid, LNA) modification has been synthesized in design.Lock Nucleotide is a kind of antisense oligonucleotide of synthetic, can with the special stable combination of target nucleic acid molecule, thereby increase the melting temperature (Tm) of probe, i.e. Tm value, specificity and stability that PCR is reacted are strengthened.The sequence of this lock nucleotide probe LNA-probe is 5'-FAM-TGTAA
t* G
g* TTGT
t* C
g* CAA-BHQ1-3'(is labeled as asterisk and the Nucleotide with underscore for lock Nucleotide).
Therefore, preferably, in the present invention, real-time fluorescence quantitative PCR detects the nucleotides sequence of fluorescent probe used and classifies 5'-TGTAA as
t* G
g* TTGT
t* C
g* CAA-BHQ1-3', is labeled as asterisk and the Nucleotide with underscore for lock Nucleotide.
In the present invention, sample is the potential vitro samples that contains bacillus cereus, as food, blood, blood products, saliva, medical treatment product or medicine etc.Because bacillus cereus is food-borne pathogens, therefore preferably sample comes from food, and if sample is food, or sample is the bacterium plate culture of food etc.It is pointed out that and detect the sample that comes from food, belong to food safety detection field.
Extract reagent used in DNA and real-time fluorescence quantitative PCR detection and instrument by those skilled in the art are grasped, and have at present the reagent of shiploads of merchandise and instrument can supply to select.For example, in the specific embodiment of the present invention, can extract thalline genomic dna with QIAamp DNA Mini Kit test kit, can carry out real-time fluorescence quantitative PCR detection with Taqman Universal PCR Master Mix II with UNG test kit and ABI7500 quantitative real time PCR Instrument.But these reagent do not provide primer and the probe of quantitative fluorescent PCR.Preferably in the present invention, the primer of real-time fluorescence quantitative PCR is murB-F and murB-R, and the sequence of murB-F is 5 '-CCTTCTTCAAGTTCAAATCTCG-3 ', and the sequence of murB-R is 5 '-GTYGTAATGACAGGTGATGGA-3 '.
The reaction process of probe method quantitative fluorescent PCR is that those skilled in the art can grasp, be generally two-step approach, each circulation is included in the sex change of 95 ° of C and the extension process of 60 ° of C, study according to the inventor, the process of most preferred real-time fluorescence quantitative PCR is 40 circulations, wherein each circulation is 95 ° of C/15sec, 60 ° of C/1min.
The result that the content of probe in the reaction system of real-time fluorescence quantitative PCR detects real-time fluorescence quantitative PCR has a certain impact.According to inventor research, the content being marked in most preferred in the reaction system of real-time fluorescence quantitative PCR is 0.1 μ mol/L.
Another aspect of the present invention is to provide the detection kit of a kind of bacillus cereus, comprises the primer for the bacillus cereus murB gene that increases.Preferably, described primer is primer murB-F (sequence is 5'-CCTTCTTCAAGTTCAAATCTCG-3') and murB-R (sequence is 5'-GTYGTAATGACAGGTGATGGA-3').
In addition, test kit provided by the present invention also can comprise lock Nucleotide fluorescent probe, and the nucleotides sequence of described fluorescent probe is classified 5'-TGTAA as
t* G
g* TTGT
t* C
g* CAA-BHQ1-3', is labeled as asterisk and the Nucleotide with underscore for lock Nucleotide.
Detection kit can also comprise extracts reagent used in DNA and/or real-time fluorescence quantitative PCR detection.These reagent can be bought by market channel, as the reagent in existing extraction DNA and/or real-time fluorescence quantitative PCR detection kit carried out to packing.
Another aspect of the present invention is to provide test kit in the present invention in the application for the preparation of detecting in the product of bacillus cereus.Described product comprises detection kit, further, can also comprise the specification sheets of recording the method that detects bacillus cereus.
Preferably, the bacillus cereus in the present invention is B.cereus.
The beneficial effect that the present invention obtains is: effective monitoring fluorescent quantitative PCR process; The species specificity that bacillus cereus detects is strong, and effectively other bacterial classification of district bacillus cereus group, makes detected result more can meet the demand of actual food product safety detection; Detection method is quick, highly sensitive, is applicable to high flux screening.
For the ease of understanding, below the accompanying drawing by concrete, embodiment are described in detail the present invention.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included in and carried out reference herein, just looks like that repeated description is excessively the same in this article for their full text.
Brief description of the drawings
Fig. 1 has shown that the real-time fluorescence quantitative PCR system in a kind of embodiment of the present invention detects the fluorescent quantitative PCR curve of positive bacteria and negative control bacterial strain.
Fig. 2 has shown the circulation ratio that the real-time fluorescence quantitative PCR system in a kind of embodiment of the present invention detects.
Embodiment
Below in conjunction with accompanying drawing in detail the preferred embodiment of one of the present invention is described in detail.
Below will invention be described by specific embodiment herein.As do not specialize part, can be according to " molecular cloning experiment guide " (third edition) (Science Press that those skilled in the art were familiar with, Beijing, China, 2005) etc. in laboratory manual and the reference quoted herein listed method implement.
1.1 main agents and instrument
The QIAamp DNA Mini Kit test kit of mentioning in below describing, purchased from Qiagen company of the U.S. (Cat.No.51304); Taqman Universal PCR Master Mix II with UNG quantitative fluorescent PCR Kit is purchased from American I nvitrogen company (Cat.No.4440038); ABI7500 quantitative real time PCR Instrument, purchased from ABI company.
1.2 the extracting of bacterial strain and DNA
Bacterial strain all can be purchased from Chinese agriculture microbial strains preservation administrative center (ACCC), Chinese medicine microbial strains preservation center (CMCC) and U.S.'s representative microbial preservation center (ATCC), and strain number is as shown in table 1.The method that cultural method can be recommended according to the above-mentioned bacterial strains unit of providing be carried out, and with reference to manufacturer explanation, extracts bacterial genomes DNA with QIAamp DNA Mini Kit test kit.
Table 1 bacillus cereus and non-bacillus cereus reference culture and the result detecting by PCR method of the present invention
* note :+represent that pcr amplification result is positive;-represent that pcr amplification result is negative
1.3 real-time fluorescence quantitative PCR reactions
PCR reaction system (PCR system is all with reference to this herein): Taqman Universal PCR Master Mix II with UNG, 10.0 μ L, primer murB-F (5 '-CCTTCTTCAAGTTCAAATCTCG-3 ') (10 μ mol/L), 1 μ L, primer murB-R (5 '-GTYGTAATGACAGGTGATGGA-3 ') (10 μ mol/L), 1 μ L, lock nucleotide probe LNA-probe (5'-FAM-TGTAAT*GG*TTGTT*CG*CAA-BHQ1-3') (2 μ mol/L), 1 μ L, ROX fluorescence correction reagent (50 ×), 0.5 μ L, ultrapure water, 4.5 μ L, add 2.0 μ L templates (, the bacterial strain DNA of above-mentioned extracting separately).In the enterprising performing PCR amplification of ABI7500 quantitative real time PCR Instrument and fluoroscopic examination, amplification condition is: 40 circulations: 95 ° of C/15sec, 68 ° of C/1min.The fluorescent quantitative PCR curve of real-time fluorescence quantitative PCR system detection positive bacteria and negative control bacterial strain as shown in Figure 1.The circulation ratio that real-time fluorescence quantitative PCR system detects as shown in Figure 2.
1.4 method specificitys
To after listed table 1 strain culturing, extract total DNA, carry out the real-time fluorescence quantitative PCR method described in 1.3 joints.Result is as shown in table 1, and all bacillus cereus bacterial strains all can draw positive detected result, and all non-bacillus cereus bacterial strain detected results are all negative.
1.5 method sensitivity
Bacillus cereus ATCC14579 is after the flat lining out of LB, picking mono-clonal is inoculated in 1mL LB liquid nutrient medium and cultivates 18 ~ 24 hours, 10 times of gradient dilutions of culture, each dilution gradient is got the total DNA of 1mL extracting, by the described real-time fluorescence quantitative PCR method of 1.3 joint.Separately respectively get 1mL and carry out plate count.
When the plate count concentration obtaining after the dilution of bacillus cereus pure growth is 10
2when CFU/mL, the Ct value that PCR detects is 38.5; And concentration is while being 10CFU/mL, Ct value is greater than 40, surmounts instrument resolving limit, detects and is limited to 10
2cFU/mL.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that those of ordinary skill in the art just can design according to the present invention make many modifications and variations without creative work.Therefore, all those skilled in the art, all should be in by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (4)
1. the detection method of bacillus cereus, is characterized in that, described method, for the object of non-medical diagnosis on disease, comprising:
1) extract the DNA in detected sample;
2) DNA step 1) being obtained, as template, carries out real-time fluorescence quantitative PCR detection;
Wherein, the target that real-time fluorescence quantitative PCR detects is bacillus cereus murB gene;
Wherein, it is primer murB-F and murB-R that real-time fluorescence quantitative PCR detects primer used, and the sequence of described murB-F is 5'-CCTTCTTCAAGTTCAAATCTCG-3'; The sequence of described murB-R is 5'-GTYGTAATGACAGGTGATGGA-3';
Wherein, the nucleotides sequence that described real-time fluorescence quantitative PCR detects fluorescent probe used is classified 5'-TGTAA as
t* G
g* TTGT
t* C
g* CAA-BHQ1-3', is labeled as asterisk and the Nucleotide with underscore for lock Nucleotide;
Wherein, the fluorescein in described fluorescent probe is FAM.
2. the method for claim 1, wherein described detected sample comes from food.
3. the detection kit of bacillus cereus, is characterized in that, described test kit comprises the primer for the bacillus cereus murB gene that increases;
Wherein, described primer is primer murB-F and murB-R, and the sequence of described murB-F is 5'-CCTTCTTCAAGTTCAAATCTCG-3', and the sequence of described murB-R is 5'-GTYGTAATGACAGGTGATGGA-3';
Wherein, described test kit also comprises lock Nucleotide fluorescent probe, and the nucleotides sequence of described fluorescent probe is classified 5'-TGTAA as
t* G
g* TTGT
t* C
g* CAA-BHQ1-3', is labeled as asterisk and the Nucleotide with underscore for lock Nucleotide;
Wherein, the fluorescein in described fluorescent probe is FAM.
4. the application of test kit as claimed in claim 3 in the product for the preparation of detection bacillus cereus.
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| CN112575097B (en) * | 2020-10-12 | 2022-05-10 | 南开大学 | Liquid phase chip for detecting bacillus cereus and application |
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| CN102453753A (en) * | 2010-10-27 | 2012-05-16 | 张文龙 | Method for detecting bacillus cereus in raw milk |
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| JPH114693A (en) * | 1997-03-24 | 1999-01-12 | Nippon Suisan Kaisha Ltd | Oligonucleotide for detection of bacillus anthracis and detection using the same |
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| KR100809547B1 (en) * | 2006-10-24 | 2008-03-04 | 경희대학교 산학협력단 | A method for identifying simultaneously bacillus cereus group bacteria using multiplex pcr |
| CN102286612A (en) * | 2010-06-18 | 2011-12-21 | 中国科学院上海生命科学研究院 | Fast detection reagent kit for pathogenic microorganism |
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| 应用实时荧光PCR检测致病性蜡样芽孢杆菌;王振国等;《生物技术通讯》;20060131;第17卷(第1期);40－42 * |
| 王振国等.应用实时荧光PCR检测致病性蜡样芽孢杆菌.《生物技术通讯》.2006,第17卷(第1期),40－42. |
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