CN111020045B - Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method - Google Patents

Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method Download PDF

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CN111020045B
CN111020045B CN202010036127.4A CN202010036127A CN111020045B CN 111020045 B CN111020045 B CN 111020045B CN 202010036127 A CN202010036127 A CN 202010036127A CN 111020045 B CN111020045 B CN 111020045B
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CN111020045A (en
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刘伟
李园园
李雪玲
贾犇
韦朝春
陆长德
李亦学
曹永梅
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Shanghai Wangwang Food Group Co ltd
SHANGHAI INDUSTRIAL TECHNOLOGY INSTITUTE
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Abstract

The invention discloses a rapid constant-temperature detection method for bacillus cereus, a primer group and application. The method comprises the following steps: extracting genome DNA from a sample to be detected; carrying out constant-temperature amplification reaction under an enzyme reaction system by taking the genome DNA as a template and taking a primer group capable of amplifying a specific sequence of the bacillus cereus as a primer; and determining whether the bacillus cereus exists in the sample to be detected by judging whether the reaction result is positive or not. The detection method has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.

Description

Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method
The application is filed on 2016, 8, 30, and has the application number of 201610780460.X and the name of the invention: the divisional application of the Chinese patent application of 'a method, a primer and a kit for rapidly detecting bacillus cereus at constant temperature'; the parent application claims the priority of the Chinese patent application with the application date of 2015, 9/2, the application number of 201510556917.4 and the name of 'method, primer and kit for rapid isothermal detection of cronobacter sakazakii'.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting bacillus cereus at constant temperature.
Background
Bacillus cereus (Bacillus cereus) is widely distributed in nature and is a common food-borne pathogenic bacterium. Clinically, food poisoning caused by bacillus cereus can be divided into two types of diarrhea type and vomiting type, which are respectively caused by diarrhea toxin and vomiting toxin generated by the bacillus cereus, and the diarrhea toxin and the vomiting toxin are sensitive to heat and trypsin, so that the bacillus cereus can be prevented; the latter is stable to heat, the activity of the protease is not interfered by pepsin, even if the expression is micro-scale, the protease is harmful to human body, and the protease is not easy to prevent and often causes fulminant food poisoning. Therefore, it is very important to prevent and detect the bacteria.
The traditional bacillus cereus detection method has the defects of long detection period, relatively complex operation and low detection efficiency, and is difficult to meet the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society. In recent years, researchers have developed detection means such as PCR with the development of nucleic acid molecule detection technology, but this method requires a special detection instrument, and is not suitable for real-time in-situ detection widely used in the basic detection department, particularly in the production line of enterprises. In order to ensure the safety of food, a rapid, simple and accurate method for detecting the bacillus cereus in the food is urgently needed.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, and BIP is composed of B1C and B2) for 6 regions of a target sequence, and completes the Nucleic acid amplification reaction by incubating for about 60min under isothermal conditions using a DNA polymerase having a strand displacement activity, and generates a macroscopic reaction byproduct, namely white magnesium pyrophosphate precipitate (see Notomi T, okayama H, masubhi H, yonekawa T, watane K, amino N, hase loop-mediated isothermal amplification of DNA, scientific Acids, JUN, 63 (12E). The technology can be completed at a constant temperature without a PCR instrument or a fluorescent quantitative PCR instrument, can judge the reaction result by naked eyes, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.
Primer design is the most critical step in LAMP technology, and the conventional method is to introduce the acknowledged specific gene of a certain organism to be detected into an online website (http:// primer explorer. Jp/e) designed by LAMP primers, and set relevant parameters to generate a primer group. Bacillus cereus contains plasmids, so the prior art such as the invention patents CN 101831493B and CN 101402997B respectively carry out the design of the Bacillus cereus LAMP primer based on the plasmids or target sequences on chromosomes. However, because of the instability of plasmids in copy number and genetic structure, the LAMP primer designed aiming at the target sequence on the chromosome of Bacillus cereus in the prior art also has the problem of insufficient primer specificity, and the problem existing in the invention patent CN 101402997B is shown in the invention table 1. That is, the Bacillus cereus detection sequence used in the prior art method is not actually specific to Bacillus cereus, that is, it is possible that Bacillus cereus is erroneously identified as Bacillus cereus. Therefore, a bacillus cereus detection method capable of ensuring specificity is urgently needed in the industry, the requirements of basic detection departments on rapidness and convenience are met, and real-time on-site detection can be conveniently carried out in an enterprise production line.
Disclosure of Invention
The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying the bacillus cereus, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a Bacillus cereus LAMP primer based on microbial genome data resources (data of 8 months and 5 days in 2013) in a GenBank database, and provides a method, a primer group and a kit for rapid isothermal amplification detection of Bacillus cereus. The detection method for detecting the bacillus cereus has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenient operation and low cost.
The invention provides a method for rapidly detecting a bacillus cereus strain, which comprises the following steps:
(1) Extracting genome DNA from a sample to be detected;
(2) Carrying out constant-temperature amplification reaction under an enzyme reaction system by taking the genome DNA as a template and taking a primer group capable of amplifying the specific base sequence of the genome of the bacillus cereus as a primer;
(3) And determining whether the bacillus cereus exists in the sample to be detected or not by judging whether the reaction result is positive or not.
The method for detecting the bacillus cereus strain at constant temperature comprises the steps of extracting genome DNA from a sample to be detected, carrying out constant-temperature amplification reaction by taking the genome DNA as a template and taking a bacillus cereus specific amplification primer group as a primer, and then determining whether the bacillus cereus exists in the sample to be detected by judging whether a reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to DNA polymerase reaction system.
In the invention, the genome-specific base sequence of the Bacillus cereus is the sequence of 3193434-3194011 bp base sequences of the Bacillus cereus with the GI number of 218895141.
In the invention, the primer group capable of amplifying the genome specific base sequence of the bacillus cereus is a part of the nucleic acid sequence of 3193434-3194011 bp positions of the genome (GI No. 218895141) or a part of a complementary strand thereof. Wherein the Bacillus cereus genome-specific nucleotide sequence is a nucleotide sequence that is unique to the Bacillus cereus genome and is not contained in the genome of another microorganism.
Wherein, the primer group capable of amplifying the specific base sequence of the bacillus cereus genome comprises but not limited to a primer group A, or a primer group with 55 percent of homology or more with a single sequence in the sequence of the primer group or the complementary strand sequence thereof.
Primer set a:
the upstream outer primer F3_ A:5 'AGGAACTATTAGAAAACGCG-containing 3' (SEQ ID NO: 1);
downstream outer primer B3_ A:5 'CCGGTTTAGATAATTCACGT-3' (SEQ ID NO: 2);
upstream inner primer FIP _ a:5 'CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAAGAAGTTC-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5-.
In the present invention, the primer set capable of amplifying the specific nucleotide sequence of the bacillus cereus genome may further include a primer set having a homology of 55% or more with a single sequence in the sequences of each of the aforementioned primer sets or the complementary strand sequences thereof, and the primer set includes, but is not limited to, the following primer sets B:
primer set B:
the upstream outer primer F3_ B:5 'TAGAAAACGCGGGTATGA-3' (SEQ ID NO: 5) (homology of 55% to the primer F3_ A5 'AGGAACTATTAGAAAACGCG-3');
the downstream outer primer B3_ B:5 'CCGGTTTAGATAATTCACGT-doped 3' (SEQ ID NO: 6);
upstream inner primer FIP _ B:5' CTAGCGCGCCTTTGTATGTTCCTCGCAGAGGTTTTTAGAAGTT-;
a downstream inner primer BIP _ B:5 'CCACAGACACAAATGTAACTCATGGTTCTCTCCTTCTTTT-containing material 3' (SEQ ID NO: 8).
In the method of the present invention, in a specific embodiment, the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer solution, 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl,10mmol/L (NH 4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in enzyme reaction system 2+ And (6) merging.
In the method of the invention, the reaction procedure of the isothermal amplification reaction is (1) incubation at 60-65 ℃ for 10-90 min, preferably 10-60 min; (2) the reaction is stopped at 80 ℃ for 2-20 min. The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis image shows a characteristic stepped strip, the sample to be detected is positive to the bacillus cereus and contains the bacillus cereus; and if the electrophoretogram does not present a characteristic stepped strip, the sample to be detected is negative to the bacillus cereus. The turbidity detection is to detect turbidity by visual observation or a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is positive to bacillus cereus and contains bacillus cereus; if no turbidity is found, the sample to be detected is negative to the bacillus cereus. Or the bottom of the reaction tube can be observed by naked eyes after centrifugation to see whether the sediment exists or not, if the sediment exists at the bottom of the reaction tube, the sample to be detected is positive to the bacillus cereus and contains the bacillus cereus; if no precipitate is formed at the bottom of the reaction tube, the sample to be detected is negative to the bacillus cereus.
The color development detection is to add a color development reagent including but not limited to calcein (50 μ M) or SYBR Green I (30-50X), or hydroxynaphthol blue (i.e. HNB,120-150 μ M) into a reaction tube. When calcein or SYBR Green I is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to bacillus cereus; if the color after the reaction is green, the sample to be detected is positive to the bacillus cereus and contains the bacillus cereus. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative to bacillus cereus; if the color after the reaction is sky blue, the sample to be detected is positive to the bacillus cereus. The chromogenic detection can be used for detecting the reaction result in real time or at the end point through a detection instrument besides observing the reaction result through naked eyes, and by reasonably setting a threshold value of negative reaction, when the reaction result of the sample to be detected is lower than or equal to the threshold value, the sample to be detected is negative to the bacillus cereus; and when the reaction result of the sample to be detected is greater than the threshold value, determining that the sample to be detected is positive to the bacillus cereus. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification microfluidic chip nucleic acid analyzer, a Genie II isothermal amplification fluorescence detection system and the like.
In the color development detection, if calcein or hydroxynaphthol blue is used as a color developing agent, the color developing agent can be added before the constant-temperature amplification reaction, or can be added after the constant-temperature amplification reaction is completed, preferably before the constant-temperature amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is adopted as the color developing agent, the SYBR Green I is added after the isothermal amplification reaction is finished. If adoptedThe calcein is used as color-developing agent, and 0.6-1mM [ Mn ] is added simultaneously with 50 μ M calcein in the enzyme reaction system 2+ ]For example, 0.6-1mM MnCl 2 。
The invention also provides a primer used in the method for detecting the bacillus cereus strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of the genome of the bacillus cereus, and the sequence of the primer comprises but is not limited to a part of a nucleic acid sequence with the length of 3193434-3194011 bp of the genome of the bacillus cereus with the GI number of 218895141 or a part of a complementary strand of the nucleic acid sequence.
Wherein the primer group capable of amplifying the specific nucleotide sequence of the Bacillus cereus genome is selected from any one of the following primer groups, or is selected from any one of the primer groups having homology of 55% or more with a single sequence in the sequences of the primer groups or the complementary strand sequence thereof. Wherein, the primer group includes but is not limited to the following primer group A. The primer set having a homology of 55% or more with a single sequence in the aforementioned primer set sequence or its complementary strand sequence includes, but is not limited to, the following primer set B.
Primer set a:
the upstream outer primer F3_ A:5 'AGGAACTATTAGAAAACGCG-doped 3';
downstream outer primer B3_ A:5 'CCGGTTTAGATAATTCACGT-3';
upstream inner primer FIP _ A:5 'CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAAGAAGTTC-3'; the downstream inner primer BIP _ A:5' CCACAGACACAAATGTAACTCATGATGTTCTCTCCTTCCT-;
primer set B:
the upstream outer primer F3_ B:5 'TAGAAAACGCGGGTATGA-3';
downstream outer primer B3_ B:5 'CCGGTTTAGATAATTCACGT-3';
upstream inner primer FIP _ B:5' CTAGCGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAAGTT-;
the downstream inner primer BIP _ B:5 'CCACAGACACAAATGTAACTCATGGTTCTCTCCCTTTCTTTT-3'.
The invention also provides a kit used in the method for detecting the bacillus cereus strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the bacillus cereus genome. In the kit of the invention, the primer group capable of amplifying the genome specific nucleotide sequence of Bacillus cereus comprises but is not limited to a part of the nucleic acid sequence of 3193434-3194011 bp site of genome (GI No. 218895141) or a part of the complementary strand thereof as the primer sequence; the primer includes but is not limited to the primer set a. But not limited to, a primer group having a homology of 55% or more with a single sequence in the aforementioned primer sequence or its complementary strand sequence as a primer; including but not limited to primer set B.
The kit also comprises Bst DNA polymerase buffer solution, bst DNA polymerase, dNTP solution and Mg 2+ (MgSO 4 Or MgCl 2 ) And betaine. In a specific embodiment, the enzyme reaction system of the kit comprises 1 XBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl,10mmol/L (NH 4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in enzyme reaction system 2+ And (6) merging.
The kit of the invention further comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, a whole genomic DNA, a partial genomic DNA of a bacillus cereus genomic DNA, or a vector comprising a whole genomic DNA or a partial genomic DNA of a bacillus cereus genomic DNA.
The kit of the invention further comprises a negative control template, and the negative control template comprises but is not limited to double distilled water.
The kit also comprises a color-developing agent, wherein the color-developing agent comprises but is not limited to calcein, SYBR Green I or hydroxynaphthol blue. When developing colorWhen the agent is calcein, the kit also contains [ Mn 2+ ]For example, mnCl 2 。
The kit of the invention also comprises double distilled water.
The kit of the invention also comprises a nucleic acid extraction reagent.
The invention also provides a carrier, which comprises any one primer selected from the primer groups A and B. The vector contains a DNA sequence with the specificity of the bacillus cereus, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application fields of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one of the primers of primer set A, vector pBR322-B containing any one of the primers of primer set B, and the like. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, and the like.
The invention also provides application of the primers selected from any one of the primer groups A and B in constant temperature detection of the bacillus cereus.
The invention also provides application of the kit in constant temperature detection of bacillus cereus.
The invention also provides application of the vector in constant temperature detection of bacillus cereus.
The invention provides a simple, rapid and sensitive method for detecting bacillus cereus, a primer/primer group and a detection reagent/kit for the technical field of food safety detection, and has great significance for food safety in China. The beneficial effects of the invention include: the bacillus cereus detection method has the advantages of strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the current common detection method, the constant temperature amplification method adopted by the invention can be carried out under the constant temperature condition, only a simple constant temperature device is needed, expensive instruments in PCR experiments are not needed, and the steps of carrying out electrophoresis detection on the amplified products and the like are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments for popularization and use, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base of molecular biology. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.
Drawings
FIG. 1 shows the specificity of the isothermal detection method for Bacillus cereus of example 7 of the present invention.
FIG. 2 shows the sensitivity of the Bacillus cereus detection method of example 8 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be determined by the appended claims. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.
EXAMPLES 1-6 Bacillus cereus isothermal reaction System and detection method
The detection is carried out according to the following steps (1) to (3):
(1) Extraction of genomic DNA
The bacillus cereus strain used for detection is from China general microbiological culture Collection center with the number of CGMCC 1.3760 (= ATCC 14579). 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co 260 /OD 280 At a concentration of 1.8, 5.36 ng/. Mu.L.
(2) Taking the genome DNA of the bacillus cereus to be detected as a template, respectively adopting self-prepared kits (shown in table 2 and table 3), preparing a reaction system according to the conditions in table 3, and taking a bacillus cereus specific amplification primer group as a primer to carry out constant-temperature amplification reaction. The primers in examples 1 to 6 were primer sets A, A, A, B, B, B, respectively.
(3) The amplification results were confirmed by electrophoresis, turbidity or color development under the conditions shown in Table 3.
As can be seen from Table 3, the detection method and the primer set and the reaction system adopted by the detection method can well amplify the specific fragment of the Bacillus cereus and obtain the detection result. Therefore, the invention can be applied to detecting whether the sample contains the bacillus cereus.
Example 7 Bacillus cereus-specific assay
Bacillus cereus 28 strains (1 to 4 in Table 4 and FIG. 1, 6 to 29) were collected, cultured separately from Bacillus cereus strains (5 in Table 4 and FIG. 1), 1mL of the culture was taken, bacterial DNA was extracted using kit IA, and LAMP amplification (primer set A) and visualization by addition of a color-developing agent were carried out, respectively, with reference to the reaction system and conditions of example 1.
The detection results are shown in Table 4 and FIG. 1, in FIG. 1, 1-4 are Staphylococcus aureus, staphylococcus aureus subspecies aureoflavus, staphylococcus epidermidis, rhodococcus equi, 6-29 are Bacillus mycoides, listeria monocytogenes, listeria inoke, listeria ehelii, salmonella enterica subspecies enterica, salmonella enteritidis, salmonella typhimurium, salmonella paratyphi B, shigella dysenteriae, shigella boydii, shigella flexneri, escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, escherichia coli diarrheal, enterotoxigenic Escherichia coli, escherichia coli hemorrhagic, yersinia enterocolitica, yersinia pseudotuberculosis, vibrio vulnificus, vibrio parahaemolyticus, vibrio franciscensis and Vibrio cereus, respectively: negative control, 5: bacillus cereus. In FIG. 1, the amplification reaction product of only the Bacillus cereus strain showed a bright green color, which is a positive result, as shown in tube No. 5. The products of other non-Bacillus cereus strains and the negative control amplification reaction are orange, and are negative results, as shown in tubes No. 1-4, no. 6-29 and NTC negative control tube.
As can be seen from the results of FIG. 1 and Table 4, the detection kit and the detection method of the present invention have good specificity for Bacillus cereus strains, i.e., only Bacillus cereus strains amplify positively, and other non-Bacillus cereus strains are negative.
Preparing a detection kit, wherein a primer adopted in the kit is a primer group B, and obtaining the same detection result according to the specific detection method, namely, the product obtained after the amplification reaction of the non-bacillus cereus strain and the negative control is a negative result, and the product obtained after the amplification reaction of the bacillus cereus strain is a positive result.
In addition, theoretical analysis was performed on the specificity of each of the primer sets a to B according to the method described in table 1, and as a result, it was found that, in the case where each primer allows three mismatches at most, at most 1 primer in each primer set was aligned to bacillus cereus, indicating that the specificity of each primer set was good.
Example 8 sensitivity detection
DNA of the bacterium CGMCC 1.3760 was extracted by the method of example 2, and the DNA was added to the reaction system using the kit IIB in a gradient of 1ng, 100pg, 10pg, 1pg, 100fg, and 10fg DNA, and LAMP amplification (primer set A) and visualization by adding a color reagent were performed under the other reaction conditions according to the method of example 2 of Table 3. As shown in FIG. 2, 1 to 6 are 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 1ng, 100pg and 10pg treatments appeared bright green and as positive results, the reaction products of 1pg, 100fg and 10fg treatments and the negative control appeared orange and as negative results. The results of the tests showed that a minimum of 10pg (about 1700 bacteria) of DNA was detected in each reaction tube.
According to the above detection method, with the same other steps and conditions as above, DNA as low as 10pg in each reaction tube can be detected using the primer set B.
TABLE 1 specificity analysis of primers in existing detection methods for Bacillus cereus
Figure BDA0002366071790000091
Note: performing Blast comparison on the detection region sequence of the primer in the patent CN 101402997B in public database resources, and determining the base numbers of the mismatch of the detection region and each primer region of the bacillus cereus and the non-bacillus cereus respectively. The lower the number of mismatched bases of the primer region and strains of Bacillus cereus (including Yersinia pseudotuberculosis and Yersinia pestis), the higher the degree of match, the poorer the specificity.
TABLE 2 kit species and main components for isothermal detection of bacillus cereus
Figure BDA0002366071790000092
TABLE 3 examples 1-6 reaction conditions and results of detection in the method for isothermal detection of Bacillus cereus according to the present invention
Figure BDA0002366071790000101
TABLE 4 strains used in the test and test results
Figure BDA0002366071790000102
Figure BDA0002366071790000111
Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china medical bacteria strain preservation and management center. b) +: positive result, -: and (5) negative result.
<110> Shanghai Industrial & technical research institute, shanghai Wangwang food group Co., ltd
<120> bacillus cereus nucleic acid rapid constant-temperature detection method and application
<160> 8
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
aggaactatt agaaaacgcg 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
ccggtttaga taattcacgt 20
<210> 3
<211> 41
<212> DNA
<213> Artificial sequence
<400> 3
ctagcgcctt tgtatgttcc tcgcagaggt tttagaagtt c 41
<210> 4
<211> 45
<212> DNA
<213> Artificial sequence
<400> 4
ccacagaaca caaatgtaac tcatgaatgt ttctctccct ttcct 45
<210> 5
<211> 18
<212> DNA
<213> Artificial sequence
<400> 5
tagaaaacgc gggtatga 18
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
ccggtttaga taattcacgt 20
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence
<400> 7
ctagcgcctt tgtatgttcc tcgcagaggt tttagaagtt 40
<210> 8
<211> 45
<212> DNA
<213> Artificial sequence
<400> 8
ccacagaaca caaatgtaac tcatggtttc tctccctttc ctttt 45

Claims (9)

1. A rapid constant-temperature detection method aiming at Bacillus cereus for non-diagnosis purposes is characterized by comprising the following steps:
(1) Extracting genome DNA from a sample to be detected;
(2) Taking the genome DNA as a template, taking a primer group capable of amplifying the specific base sequence of the genome of the bacillus cereus as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system;
(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;
wherein the bacillus cereus genome specific base sequence is 3193434-3194011 bp bit sequence of bacillus cereus genome with GI number 218895141;
wherein the primer group capable of amplifying the specific base sequence of the bacillus cereus genome is a primer group B;
primer set B:
the upstream outer primer F3_ B:5 'TAGAAAACGCGGGTATGA-3';
downstream outer primer B3_ B:5 'CCGGTTTAGATAATTCACGT-3';
upstream inner primer FIP _ B:5' CTAGCGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAAGTT-;
a downstream inner primer BIP _ B:5 'CCACAGACACAAATGTAACTCATGGTTCTCTCCCTTTCTTTT-3'.
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP,0.8-2.0 mu mol/L FIP _ B and BIP _ B primers, 0.15-0.3 mu mol/L F3_ B and B3_ B primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: (1) incubating for 10-90 min at 60-65 ℃; (2) the reaction is terminated for 2-20 min at 80 ℃.
4. The primer for rapid constant temperature detection of the bacillus cereus is characterized by comprising a primer group capable of amplifying a specific base sequence of a bacillus cereus genome, wherein the specific base sequence of the bacillus cereus genome is a part of a nucleic acid sequence or a part of a complementary strand of the nucleic acid sequence at 3193434-3194011 bp site of the bacillus cereus genome with the GI number of 218895141;
wherein the primer group capable of amplifying the specific base sequence of the bacillus cereus genome is a primer group B;
primer set B:
the upstream outer primer F3_ B:5 'TAGAAAACGCGGGTATGA-3';
downstream outer primer B3_ B:5 'CCGGTTTAGATAATTCACGT-3';
upstream inner primer FIP _ B:5' CTAGCGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAAGTT-;
the downstream inner primer BIP _ B:5 'CCACAGACACAAATGTAACTCATGGTTCTCTCCTTCTTTT-containing material 3'.
5. A rapid isothermal detection kit for Bacillus cereus, comprising the primer according to claim 4.
6. The kit of claim 5, further comprising a Bst DNA polymerase reaction buffer, bst DNA polymerase, dNTP solution, mg 2+ And one or more of betaine.
7. The kit of claim 5, wherein the enzymatic reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer solution, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP,0.8-2.0 μmol/L FIP _ B and BIP _ B primers, 0.15-0.3 μmol/L F3_ B and B3_ B primers, 0.16-0.64U/μ L Bst DNA polymerase, and 0-1.5mol/L betaine.
8. Use of a primer for isothermal detection of bacillus cereus for non-diagnostic purposes, wherein the primer is according to claim 4.
9. Use of a kit according to any one of claims 5 to 7 for the isothermal detection of Bacillus cereus for non-diagnostic purposes.
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