CN104032000B - The detection method of a kind of bacillus cereus and test kit - Google Patents
The detection method of a kind of bacillus cereus and test kit Download PDFInfo
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- 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
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
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Abstract
The detection method providing a kind of new bacillus cereus of the present invention, this detection method includes: 1) extract the DNA in detected sample；2) using step 1) DNA that obtains as template, carry out real-time fluorescence quantitative PCR detection；Wherein, the target of real-time fluorescence quantitative PCR detection is bacillus cereus murB gene.The present invention also provides for the detection kit of a kind of bacillus cereus, and described test kit includes the primer for expanding bacillus cereus murB gene.
Description
Technical Field
The invention belongs to nucleic acid detection in the field of food safety. In particular, the invention relates to a method and a kit for rapidly detecting bacillus cereus.
Background
Bacillus cereus (Bacillus cereus) is one of aerobic Bacillus groups, non-capsular, motile, gram-positive, and spore-tolerant for 30 minutes at 100 ℃. The strain is widely distributed in nature and is commonly found in soil, sewage and dust; can grow and propagate at 16-45 deg.c, and has optimal growth temperature of 35 deg.c. Food poisoning is often caused by the bacteria contaminating and propagating food products such as starch products rich in carbohydrates and dairy products rich in proteins. Bacillus cereus is also a conditional pathogen of various parenteral infectious diseases of human and animals, and can cause diseases of conjunctivitis, respiratory system, cardiovascular system, central nervous system, wound infection and the like of human. The main pathogenic substances of the bacillus cereus are heat-resistant enterotoxin and heat-labile enterotoxin which cause human diseases, a large number of live bacteria and metabolites such as phospholipase, nuclease, hemolysin and the like. In recent years, there have been increasing cases of Bacillus cereus as a wound-infecting bacterium on the skin. It has been found that the burn wound infection symptoms also cause gas gangrene, develop rapidly, and muscle necrosis occurs. Systemic toxicosis symptoms are caused by exotoxins of the bacteria. Such bacterial infections, are not effective against any antibiotic.
In food enterprises, especially grain and oil processing enterprises, serious pollution control problems of bacillus cereus are faced, however, a serious problem in the prior art is that the detection, especially the quantitative detection, of the bacillus cereus is very difficult, so that the prevention and the treatment of the biohazard factor become empty.
An effective quantitative detection method for food-borne bacillus cereus is established to improve the detection speed and accuracy of the bacillus cereus, so that polluted food is treated in time, and the method has important significance in public health, food sanitation, port quarantine and the like.
However, the traditional detection method is time-consuming and tedious in operation, and products based on the immunological principle have the problems of high detection limit, low specificity and the like. Especially 6 species of the bacillus cereus group, including bacillus cereus, bacillus anthracis, bacillus thuringiensis, bacillus mycoides, bacillus pseudomycoides and bacillus westernephiensis, have very high similarity in phenotypic and biochemical characteristics, and only differ in enterotoxigenic species, presence or absence of parasporal crystals, especially the presence of some variant strains that do not produce enterotoxigenic or carry parasporal crystals, making identification of the group very difficult.
Therefore, through long-term research, the inventor surprisingly researches a method for fluorescence quantitative rapid detection of locked nucleotide probe with bacillus cereus murB as target, which not only can effectively monitor PCR amplification, but also has strong species specificity of bacillus cereus, can effectively distinguish other bacillus of bacillus cereus flora, is rapid and high in detection sensitivity, and can be applied to high-throughput screening of bacillus cereus in food.
Disclosure of Invention
The first aspect of the invention provides a novel detection method of bacillus cereus. The detection method comprises the following steps: 1) extracting DNA in a sample to be detected; 2) performing real-time fluorescent quantitative PCR detection by using the DNA obtained in the step 1) as a template; wherein, the target of the real-time fluorescence quantitative PCR detection is the murB gene of the bacillus cereus.
The invention takes the murB gene as the detection target and is obtained by the following screening method:
the download collection was searched through a common database to create a gene library for Bacillus cereus and Bacillus cereus that were not waxy. A Bacillus cereus genome is selected as a target genome, and is segmented by a program genome cut.pl, wherein the segment size is 100 bp. The similarity analysis of the divided fragments with other B.cereus genomes in the species was performed by BLAST, and conserved fragments (i.e., fragments contained in each B.cereus genome) in the species were discovered by the program cofeder. And performing similarity analysis on the conserved segment and other non-bacillus cereus genomes outside the species by using BLAST, and discovering species specific segments (the judgment standard of the species specific segments, namely the homologous regions of the conserved segment in the species and all other sequenced microorganism genomes are not more than 25bp) by using a program spfinder.
A genome library is established by using a genomics method, and then 100 specific fragments, namely 100 specific DNA sequence fragments with the size of 100bp, are screened out from the target bacillus cereus genome through online BLAST. The 100 specific fragments are merged and arranged into 87 fragments, primers are respectively designed to carry out common PCR detection evaluation of specificity and sensitivity, and finally, the specificity and the sensitivity of the primers corresponding to the murB gene are proved to be optimal, so that the murB gene is selected as a detection target.
In order to distinguish several strains of the bacillus cereus group, probes modified by Locked Nucleotide (LNA) added to the conserved site specific to the murB gene are designed and synthesized through analysis of the conserved site of the murB gene. The locked nucleotide is an artificial antisense oligonucleotide, which can be specifically and stably combined with a target nucleic acid molecule, thereby increasing the melting temperature, namely the Tm value, of the probe and enhancing the specificity and stability of the PCR reaction. The sequence of the locked nucleotide probe LNA-probe is 5' -FAM-TGTAAT*GG*TTGTT*CGCAA-BHQ1-3' (marked with an asterisk and underlined nucleotides are locked nucleotides).
Therefore, preferably, in the present invention, the nucleotide sequence of the fluorescent probe used for real-time fluorescent quantitative PCR detection is 5' -TGTAAT*GG*TTGTT*CGCAA-BHQ1-3', marked with an asterisk and underlined nucleotides are locked nucleotides.
In the present invention, the sample is an isolated sample potentially containing Bacillus cereus, such as food, blood products, saliva, medical supplies or drugs, etc. Since Bacillus cereus is a food-borne pathogen, it is preferred that the sample is derived from a food product, such as a food product, or a bacterial plate culture of a food product. It is noted that the detection of samples from food products is in the field of food safety detection.
Reagents and instruments used in DNA extraction and real-time fluorescent quantitative PCR detection are well known to those skilled in the art, and a large number of commercially available reagents and instruments are currently available. For example, in the embodiment of the present invention, the genomic DNA of the cells can be extracted using QIAamp DNA Mini Kit, and real-time fluorescent quantitative PCR can be detected using Taqman Universal PCR Master Mix II with UNG Kit and ABI7500 fluorescent quantitative PCR instrument. However, these reagents do not provide primers and probes for fluorescent quantitative PCR. Preferably, in the present invention, primers for real-time fluorescent quantitative PCR are murB-F and murB-R, the sequence of murB-F is 5'-CCTTCTTCAAGTTCAAATCTCG-3', and the sequence of murB-R is 5 '-GTYGTAATGACAGGTGATGGA-3'.
The reaction process of the probe-based quantitative fluorescence PCR is well known to those skilled in the art, and is generally a two-step process, each cycle comprising denaturation at 95 ℃ and extension at 60 ℃, and according to the study of the present inventors, the most preferable process of the real-time quantitative fluorescence PCR is 40 cycles, wherein each cycle is 95 ℃/15sec and 60 ℃/1 min.
The content of the probe in a reaction system of the real-time fluorescent quantitative PCR has certain influence on the result of the real-time fluorescent quantitative PCR detection. According to the research of the inventor, the most preferable content of the internal standard in the reaction system of the real-time fluorescence quantitative PCR is 0.1 mu mol/L.
Another aspect of the present invention provides a detection kit for Bacillus cereus, comprising primers for amplifying the MurB gene of Bacillus cereus. Preferably, the primers are primers murB-F (sequence 5'-CCTTCTTCAAGTTCAAATCTCG-3') and murB-R (sequence 5 '-GTYGTAATGACAGGTGATGGA-3').
In addition, the kit provided by the invention can also comprise a locked nucleotide fluorescent probe, and the nucleotide sequence of the fluorescent probe is 5' -TGTAAT*GG*TTGTT*CGCAA-BHQ1-3', marked with an asterisk and underlined nucleotidesLocked nucleotides.
The test kit may also include reagents used in DNA extraction and/or real-time fluorescent quantitative PCR tests. These reagents can be purchased from commercial sources, for example, reagents in existing DNA extraction and/or real-time fluorescent quantitative PCR detection kits can be dispensed.
In a further aspect, the invention provides the use of a kit of the invention in the manufacture of a product for the detection of bacillus cereus. The product comprises a detection kit and further comprises instructions for recording a method for detecting the bacillus cereus.
Preferably, the bacillus cereus in the present invention is b.
The invention has the following beneficial effects: the fluorescent quantitative PCR amplification process is effectively monitored; the species specificity of bacillus cereus detection is strong, and other strains of bacillus cereus flora can be effectively distinguished, so that the detection result can better meet the requirement of actual food safety detection; the detection method is rapid, high in sensitivity and suitable for high-throughput screening.
For the sake of understanding, the present invention will be described in detail below with reference to specific drawings and examples. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced in their entirety.
Drawings
FIG. 1 shows the fluorescence quantitative PCR amplification curve of the real-time fluorescence quantitative PCR system for detecting the positive bacteria and the negative control strain in one embodiment of the invention.
FIG. 2 shows the reproducibility of the detection of the real-time fluorescent quantitative PCR system in one embodiment of the present invention.
Detailed Description
A preferred embodiment of the present invention is described in detail below with reference to the accompanying drawings.
The invention will be described herein below by means of specific examples. Unless otherwise specified, the method can be performed according to the methods listed in the experimental manuals such as "molecular cloning laboratory Manual" (third edition) (scientific Press, Beijing, China, 2005) and the like, which are familiar to those skilled in the art, and the references cited therein.
1.1 Primary reagents and instruments
The QIAamp DNA Mini Kit referred to in the description below was purchased from Qiagen, USA (Cat. No. 51304); taqman Universal PCR Master Mix II with UNG fluorescent quantitative PCR Kit was purchased from Invitrogen USA (Cat. No. 4440038); ABI7500 fluorescent quantitative PCR instrument, purchased from ABI.
1.2 Strain and extraction of DNA
The strains can be purchased from China agricultural microbial culture Collection center (ACCC), China medical microbial culture Collection center (CMCC) and American Type Culture Collection (ATCC), and the strain numbers are shown in Table 1. The culture method can be carried out according to the method recommended by the above-mentioned strain providing unit, and the bacterial genomic DNA can be extracted using QIAamp DNA Mini Kit according to the manufacturer's instructions. TABLE 1 Bacillus cereus and Bacillus cereus Standard strains and the results of detection by the PCR method of the present invention
Injecting: + represents that the PCR amplification result is positive; negative result of PCR amplification
1.3 real-time fluorescent quantitative PCR reaction
PCR reaction system (reference is made to PCR systems herein): taqman Universal PCR Master Mix IIwith UNG, 10.0. mu.L, primer murB-F (5'-CCTTCTTCAAGTTCAAATCTCG-3') (10. mu. mol/L), 1. mu.L, primer murB-R (5 '-GTYGTAATGACAGGTGATGGA-3') (10. mu. mol/L), 1. mu.L, locked nucleotide probe LNA-probe (5'-FAM-TGTAAT GG TTGTT CG CAA BHQ1-3') (2. mu. mol/L), 1. mu.L, ROX fluorescence correction reagent (50X), 0.5. mu.L, ultrapure water, 4.5. mu.L; mu.L of template (i.e., the DNA of each of the above-mentioned strains extracted) was added. Performing PCR amplification and fluorescence detection on an ABI7500 fluorescence quantitative PCR instrument, wherein the amplification conditions are as follows: 40 cycles: 95 ℃/15sec,68 ℃/1 min. The fluorescent quantitative PCR amplification curve of the real-time fluorescent quantitative PCR system for detecting the positive bacteria and the negative control strain is shown in figure 1. The reproducibility of the real-time fluorescent quantitative PCR system is shown in FIG. 2.
1.4 method specificity
Total DNA was extracted from the cultured strains listed in Table 1 and subjected to the real-time fluorescent quantitative PCR method described in section 1.3. As shown in Table 1, all Bacillus cereus strains gave positive results, while all non-Bacillus cereus strains gave negative results.
1.5 method sensitivity
After streaking the bacillus cereus ATCC14579 on an LB plate, selecting a single clone, inoculating the single clone in 1mL of LB liquid culture medium, culturing for 18-24 hours, diluting the culture by 10-fold gradient, extracting 1mL of total DNA from each dilution gradient, and performing real-time fluorescence quantitative PCR according to the 1.3 section. Another 1mL portion was counted on the plate.
The plate count concentration obtained when the pure culture of Bacillus cereus was diluted was 102CFU/mL timeThe Ct value detected by PCR is 38.5; when the concentration is 10CFU/mL, the Ct value is more than 40 and exceeds the resolution limit of the instrument, namely the detection limit is 102CFU/mL。
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (4)
1. A method for detecting bacillus cereus, said method being for non-disease diagnostic purposes, comprising:
1) extracting DNA in a sample to be detected;
2) performing real-time fluorescent quantitative PCR detection by using the DNA obtained in the step 1) as a template;
wherein, the target of the real-time fluorescence quantitative PCR detection is a bacillus cereus murB gene;
the nucleotide sequence of the fluorescent probe used for real-time fluorescent quantitative PCR detection is 5' -TGTAAT*GG*TTGTT*CGCAA-BHQ1-3', marked with an asterisk and underlined nucleotides are locked nucleotides; primers used for real-time fluorescent quantitative PCR detection are primers murB-F and murB-R; wherein,
the sequence of murB-F is 5'-CCTTCTTCAAGTTCAAATCTCG-3';
the sequence of murB-R is 5 '-GTYGTAATGACAGGTGATGGA-3'.
2. The method of claim 1, wherein the sample to be tested is derived from a food product.
3. The detection kit for the bacillus cereus is characterized by comprising a primer for amplifying the bacillus cereus murB gene; wherein the primers are primers murB-F and murB-R; wherein,
the sequence of murB-F is 5'-CCTTCTTCAAGTTCAAATCTCG-3';
the sequence of murB-R is 5 '-GTYGTAATGACAGGTGATGGA-3';
the kit also comprises a locked nucleotide fluorescent probe, and the nucleotide sequence of the fluorescent probe is 5' -TGTAAT*GG*TTGTT*CGCAA-BHQ1-3', marked with an asterisk and underlined nucleotides are locked nucleotides.
4. Use of the kit of claim 3 in the preparation of a product for detecting bacillus cereus.
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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 |
| CN101928773A (en) * | 2010-05-14 | 2010-12-29 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Oligonucleotide primer for detecting common pathogenic bacteria by adopting fluorescent quantitation PCR (Rich Client Platform) technology, method thereof for detecting common pathogenic bacteria and application thereof |
| CN102453753A (en) * | 2010-10-27 | 2012-05-16 | 张文龙 | Method for detecting bacillus cereus in raw milk |
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