CN103710456A - Primer for identifying whether bacterial strain of aspergillus flavus generates aflatoxin and application thereof - Google Patents
Primer for identifying whether bacterial strain of aspergillus flavus generates aflatoxin and application thereof Download PDFInfo
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- CN103710456A CN103710456A CN201410002187.9A CN201410002187A CN103710456A CN 103710456 A CN103710456 A CN 103710456A CN 201410002187 A CN201410002187 A CN 201410002187A CN 103710456 A CN103710456 A CN 103710456A
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Images
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses a primer for identifying whether bacterial strain of aspergillus flavus generates aflatoxin or not and application of the primer. The primer for identifying the bacterial strain of aspergillus flavus not generating aflatoxin comprises the following four primer pairs: a primer pair 1 formed by two single-stranded DNA shown by a sequence 1 and a sequence 2 in a sequence table, a primer pair 2 formed by two single-stranded DNA shown by a sequence 3 and a sequence 4 in the sequence table, a primer pair 3 formed by two single-stranded DNA shown by a sequence 5 and a sequence 6 in the sequence table, and a primer pair 4 formed by two single-stranded DNA shown by a sequence 7 and a sequence 8 in the sequence table. Experiments show that the primer for identifying whether bacterial strain of aspergillus flavus generates aflatoxin or not has high sequence specificity, has the characteristics of high speed and high reliability by PCR (polymerase chain reaction) amplification identification, provides a good technological manner for screening the bacterial strain of aspergillus flavus not generating aflatoxin rapidly and accurately at high flux, and has importance in controlling contamination of aflatoxin in agriculture products.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of primer and application of whether producing aflatoxin for the identification of aspergillus flavus strain.
Background technology
Aflatoxin (Aflatoxins) is that a class is mainly by mycetogenetic secondary metabolites such as flavus (Aspergillus flavus) and Aspergillus parasiticuses (Aspergillus parasiticus), in China, aflatoxin is mainly produced by flavus.That aflatoxin has is carcinogenic, teratogenesis, cause " three cause " effect of cell mutation, only 0.294mg/kg dosage just can cause that the acute poisoning of Sensitivity animal is dead, its main action target is liver, it is one of principal element of bringing out malignant tumour primary hepatocellular carcinoma (Hepatocellular carcinoma), in the shortest 24 weeks, just hepatic necrosis canceration etc. can be caused, kidney and adrenal acute pathology can be caused in addition.Therefore, effective prevention and control aflatoxin contamination, for ensureing China's food safety and safeguarding that national economic interest is significant.
Different aspergillus flavus strains have very big-difference in product aflatoxin ability, wherein do not produce malicious flavus and can account for 0%-80%.Recently, thus utilize and not produce the content that growth that malicious flavus suppresses to produce malicious flavus controls aflatoxin in agricultural-food and obtained good application in countries and regions such as the U.S., Africa.Visible, how to identify that whether flavus produces poison is very important.At present, whether flavus is produced to poison is mainly first to cultivate aspergillus flavus strain, and then extracts toxin, and last contratoxin content detects, and wastes time and energy very much.Therefore, develop a kind of Rapid identification flavus and whether produce malicious method and do not produce malicious flavus for screening, thereby it is significant to control the pollution of aflatoxin in agricultural-food.
Summary of the invention
The object of this invention is to provide a kind of primer and application of whether producing aflatoxin for the identification of aspergillus flavus strain.
Provided by the present invention for the identification of or the assistant identification aspergillus flavus strain to be measured primer pair group of whether producing aflatoxin, specifically by following 4 primer pairs, formed: the primer pair 3 that in the primer pair 2 that in the primer pair 1 that in sequence table, two single stranded DNAs shown in sequence 1 and sequence 2 form, sequence table, two single stranded DNAs shown in sequence 3 and sequence 4 form, sequence table, two single stranded DNAs shown in sequence 5 and sequence 6 form, and the primer pair 4 that in sequence table, two single stranded DNAs shown in sequence 7 and sequence 8 form.
Aflatoxin is mainly by 29 genes in a 70kb left and right gene cluster in No. 3 karyomit(e)s of flavus, to participate in it synthesize and regulate and control.Research shows not produce malicious flavus mainly because its toxin synthetic gene disappearance causes.Wherein, described primer pair 1 is specific to aflT gene; Described primer pair 2 is specific to nor-1 gene; Described primer pair 3 is specific to aflR gene; Described primer pair 4 is specific to hypB gene.
In described primer pair group, two primers of each primer pair are used at PCR reaction system moderate.
The test kit that contains described primer pair group also belongs to protection scope of the present invention.
Another object of the present invention is to provide the preparation method of described primer pair group.
The preparation method of described primer pair group, specifically can comprise the step that described two single stranded DNAs of each primer pair in described primer pair group are packed separately respectively.
Also object of the present invention is to provide the preparation method of described test kit.
The preparation method of described test kit, specifically can comprise the steps: described two single stranded DNAs of each primer pair in described primer pair group respectively separately after packing, be packaged in same reagent box with at least one in following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
Described primer pair group; or described test kit identifying or whether assistant identification aspergillus flavus strain to be measured produces the application in aflatoxin, or also belonging to protection scope of the present invention for the preparation of the application of identifying or whether assistant identification aspergillus flavus strain to be measured produces in the product of aflatoxin.
A further object of the present invention is to provide a kind ofly utilizes described primer pair group, or the method that described test kit is identified or whether assistant identification aspergillus flavus strain to be measured produces aflatoxin.
The method specifically can comprise the steps:
(1) from aspergillus flavus strain to be measured, extract genomic dna as template, adopt 4 primer pairs in described primer pair group to carry out respectively pcr amplification, obtain 4 parts of PCR products;
(2) according to 4 parts of PCR products of step (1) gained, determine as follows whether aspergillus flavus strain to be measured produces aflatoxin: if the number of target DNA fragment is below 3 in described 4 parts of PCR products, described aspergillus flavus strain aspergillus flavus not producing toxin to be measured, or candidate's aspergillus flavus not producing toxin; If the number of target DNA fragment is 4 in described 4 parts of PCR products, described aspergillus flavus strain to be measured produces aflatoxin, or candidate produces aflatoxin;
Described target DNA fragment is for arbitrary as follows: the DNA fragmentation that the size of utilizing described primer pair 1 amplification to obtain is 1141bp; The DNA fragmentation that the size of utilizing described primer pair 2 amplifications to obtain is 977bp; The DNA fragmentation that the size of utilizing described primer pair 3 amplifications to obtain is 629bp; The DNA fragmentation that the size of utilizing described primer pair 4 amplifications to obtain is 422bp.
In step (1), while adopting described 4 primer pairs to carry out pcr amplification respectively, the annealing temperature of employing is 55 ℃.
In the step (1) of described method, the reaction conditions that carries out described pcr amplification is specially: 95 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min30s, 30 circulations; Last 72 ℃ are extended 7min.
In the step (1) of described method, in carrying out each reaction system of described pcr amplification, in described primer pair, the final concentration of every single strand dna all can be 0.2-0.5 μ M, concrete as 0.5 μ M.
Further, the reaction system of carrying out described pcr amplification is specially: template 1 μ L, each 1 μ L(final concentration of the upstream and downstream primer of 10 μ M is 0.5 μ M), 2 * Colorless
reaction Buffer10 μ L, mends distilled water 7 μ L to 20 μ L systems.Wherein, 2 * Colorless
reaction Buffer Gotag is U.S. promega company product, and its catalog number is M7132.
In described method, the nucleotide sequence of the DNA fragmentation that described size is 1141bp is specially in sequence table shown in sequence 9; Described size is that the nucleotide sequence of the DNA fragmentation of 977bp is specially in sequence table shown in sequence 10; Described size is that the nucleotide sequence of the DNA fragmentation of 629bp is specially in sequence table shown in sequence 11; Described size is that the nucleotide sequence of the DNA fragmentation of 422bp is specially in sequence table shown in sequence 12.
In the present invention, described aspergillus flavus strain to be measured is specially as lower any: Aspergillus flavus (Aspergillus flavus) DW-12, Aspergillus flavus (Aspergillus flavus) DW-14, Aspergillus flavus (Aspergillus flavus) XinZ-11, Aspergillus flavus (Aspergillus flavus) XinZ-27, Aspergillus flavus (Aspergillus flavus) XinZ-33, Aspergillus flavus (Aspergillus flavus) YC-19, Aspergillus flavus (Aspergillus flavus) HG-14, Aspergillus flavus (Aspergillus flavus) QD-1, Aspergillus flavus (Aspergillus flavus) JZ-2, Aspergillus flavus (Aspergillus flavus) GZ-17, Aspergillus flavus (Aspergillus flavus) DW-5, Aspergillus flavus (Aspergillus flavus) DW-6, Aspergillus flavus (Aspergillus flavus) XinZ-15, Aspergillus flavus (Aspergillus flavus) XinZ-24, Aspergillus flavus (Aspergillus flavus) YC-10, Aspergillus flavus (Aspergillus flavus) HG-12, Aspergillus flavus (Aspergillus flavus) HG-24, Aspergillus flavus (Aspergillus flavus) QD-15, Aspergillus flavus (Aspergillus flavus) FX-1 and Aspergillus flavus (Aspergillus flavus) GZ-9.
In the present invention, above all described aflatoxin all can be as lower at least one: AFB
1, AFB
2, AFG
1, AFG
2.
Experimental results show that, the invention provides for the identification of aspergillus flavus strain and whether produce malicious primer sequence, these sequence-specifics are high, by utilizing this primer sequence pcr amplification to identify, produce malicious flavus and do not produce malicious flavus, there is speed fast, confidence level degree high, for fast, accurately, high flux screening does not produce malicious aspergillus flavus strain that good technique means is provided, to controlling the pollution tool of aflatoxin in agricultural-food, be of great significance.
Accompanying drawing explanation
Fig. 1 is the gel electrophoresis spectrum of each bacterial strain pcr amplification product of specific detection.
Fig. 2 is AFB
1, B
2, G
1, G
2the HPLC collection of illustrative plates of hybrid standard product.Wherein, 1 is AFG in aflatoxin standard substance
2, retention time is 7.861min; 2 is AFG in aflatoxin standard substance
1, retention time is 8.793min; 3 is AFB in aflatoxin standard substance
2, retention time is 10.293min; 4 is AFB in aflatoxin standard substance
1, retention time is 11.735min.
Fig. 3 is not for producing the HPLC collection of illustrative plates of malicious aspergillus flavus strain DW-12 ferment filtrate.
Fig. 4 is for producing the HPLC collection of illustrative plates of malicious aspergillus flavus strain DW-5 ferment filtrate.
Fig. 5 is not for producing the C3 of malicious aspergillus flavus strain DW-12, XinZ-11, XinZ-27 and QD-1, aflT, the gel electrophoresis spectrum of norA and tetra-gene PCR amplified productions of hypA.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12, DW-14, XinZ-11, XinZ-27, XinZ-33, YC-19, HG-14, QD-1, JZ-2, GZ-17, and DW-5, DW-6, XinZ-15, XinZ-24, YC-10, HG-12, HG-24, QD-15, FX-1, GZ-9: be recorded in and " affix one's name to Aspergillus flavus distribution in four ecotope peanut soil of .2013. China at the beginning of, produce malicious feature and genetic diversity Journal of Sex Research [D] doctorate paper. Beijing: Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc " literary composition, public Ke Cong Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sc obtains.
AFB
1, B
2, G
1, G
2hybrid standard product (LB96951): be purchased from SUPELCO company.
According to toxin synthetic gene aflT, nor-1, the sequence of aflR and hypB, designs 4 groups of PCR primers, is respectively aflT-F/aflT-R, nor-1-F/nor-1-R, aflR-F/aflR-R and hypB-F/hypB-R.Specifically as shown in table 1.Primer is synthetic by the raw work in Shanghai, also can be synthetic by conventional primer synthetic method.
The different toxin synthetic gene of table 1 primer sequence
Whether embodiment 2, aspergillus flavus strain produce malicious evaluation
Strains tested: Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12, DW-14, XinZ-11, XinZ-27, XinZ-33, YC-19, HG-14, QD-1, JZ-2, GZ-17, and DW-5, DW-6, XinZ-15, XinZ-24, YC-10, HG-12, HG-24, QD-15, FX-1, GZ-9.
One, bacterial strain DNA extraction
With the mycelia of inoculating needle scraping 0.2g from PDA flat board, put into centrifuge tube, pour liquid nitrogen into, be then put in high-speed tissue mashing machine, mycelium is ground into powder.Mycelium powder after grinding is moved in 2mL centrifuge tube, extracting solution (the formula: solvent is water that adds 750 μ L, solute and concentration thereof are: 100mmol/L Tris-HCl, 150mmol/L EDTA, pH8.0), 50 ℃ of standing 2min, add again 150 μ L10%(10g/100ml) the SDS aqueous solution, after mixing, add 450 μ L Benzyl Chlorides, thermal agitation centrifuge tube, makes to manage interior mixture and becomes emulsus.Put in 50 ℃ of water-baths and be incubated 1h.Add the NaAc aqueous solution that 450 μ L concentration are 3mol/L, mix rear ice bath 15min.4 ℃, after the centrifugal 10min of 12000r/min, collect supernatant liquor to 1.5mL centrifuge tube.Add equal-volume chloroform: primary isoamyl alcohol (24: 1) (v/v), after fully mixing, 4 ℃, the centrifugal 5min of 12000r/min, sucking-off supernatant liquor is to the new centrifuge tube of 1.5mL, add equal-volume isopropanol precipitating 30min left and right, 4 ℃, the centrifugal 10min of 12000r/min, precipitation 70%(volume fraction) washing with alcohol, room temperature is placed dry 10min, is dissolved in the TE damping fluid of 100 μ L, and-20 ℃ save backup.Each DNA for examination aspergillus flavus strain all adopts aforesaid method to extract.
Two, primer extension method provided by the present invention identifies for examination aspergillus flavus strain whether produce poison
1, working method and result decision method
The DNA of aspergillus flavus strain to be measured of take is template, with 4 groups of synthetic primer aflT-F/aflT-R of design in embodiment 1, nor-1-F/nor-1-R, aflR-F/aflR-R and hypB-F/hypB-R carry out respectively PCR reaction, and negative control (usining sterilized water as template) is all established in each reaction.
PCR reaction system is: 1 μ L DNA profiling, concentration is that each 1 μ L(upstream and downstream primer final concentration in reaction system of upstream and downstream primer of 10 μ M is 0.5 μ M), 2 * Colorless
reaction Buffer Gotag10 μ L, distilled water complements to 20 μ L.Wherein, 2 * Colorless
reaction Buffer Gotag is U.S. promega company product, and its catalog number is M7132.
Reaction conditions is: 95 ℃ of denaturation 2min; 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min30s, carry out 30 circulations; Last 72 ℃ are extended 7min.
PCR product after electrophoresis, is taken pictures with gel imaging system in 1 * TAE damping fluid with 1.0% sepharose.
Result decision method:
If the number of target DNA fragment is below 3 in 4 parts of PCR products of the pcr amplification gained of step 1, judge described aspergillus flavus strain aspergillus flavus not producing toxin to be measured; If the number of target DNA fragment is 4 in described 4 parts of PCR products, judge that described aspergillus flavus strain to be measured produces aflatoxin.
Wherein, described target DNA fragment is for arbitrary as follows: the DNA fragmentation (sequence 9) that the size of utilizing described primer pair aflT-F/aflT-R amplification to obtain is 1141bp; The DNA fragmentation (sequence 10) that the size of utilizing described primer pair nor-1-F/nor-1-R amplification to obtain is 977bp; The DNA fragmentation (sequence 11) that the size of utilizing described primer pair aflR-F/aflR-R amplification to obtain is 629bp; The DNA fragmentation (sequence 12) that the size of utilizing described primer pair hypB-F/hypB-R amplification to obtain is 422bp.
2, to whether producing malicious evaluation for examination aspergillus flavus strain
What the step 1 of take was respectively extracted is respectively template for the DNA that tries aspergillus flavus strain, carries out pcr amplification, and according to the result decision method described in step 1, amplification is judged with reference to the method described in step 1.Experiment in triplicate.
As shown in Figure 1, Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12 and QD-1 have 3 electrophoretic bands, i.e. 1141bp(aflT to electrophoresis result), 977bp(nor-1) and 629bp(aflR), disappearance toxin synthetic gene hypB is described; Strain X inZ-11 and XinZ-27 have 2 electrophoretic bands, i.e. 1141bp(aflT) and 977bp(nor-1), disappearance toxin synthetic gene aflR and hypB are described; Bacterial strain YC-19 has 2 electrophoretic bands, i.e. 977bp(nor-1) and 422bp(hypB), disappearance toxin synthetic gene aflT and aflR are described; Bacterial strain DW-14 only has 1 electrophoretic band, i.e. 629bp(aflR), disappearance toxin synthetic gene aflT is described, nor-1 and hypB; Strain X inZ-33 and JZ-2 only have 1 electrophoretic band, i.e. 422bp(hypB), disappearance toxin synthetic gene aflT is described, nor-1 and aflR; Bacterial strain HG-14 and GZ-17 do not have electrophoretic band, illustrate that above-mentioned 4 toxin synthetic genes all lack; Therefore, judge that Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12, DW-14, XinZ-11, XinZ-27, XinZ-33, YC-19, HG-14, QD-1, JZ-2 and GZ-17 are as not producing malicious flavus.Aspergillus flavus (Aspergillus flavus) bacterial strain DW-5, DW-6, XinZ-15, XinZ-24, YC-10, HG-12, HG-24, QD-15, FX-1 and GZ-9 have 4 bands, show that they are very likely to produce malicious flavus.
Further, the present inventor reclaims order-checking to the corresponding object band of each strains tested, result demonstration, and the nucleotide sequence of object band 1141bp(aflT) is as shown in sequence in sequence table 9; The nucleotide sequence of object band 977bp(nor-1) is as shown in sequence in sequence table 10; The nucleotide sequence of object band 629bp(aflR) is as shown in sequence in sequence table 11; The nucleotide sequence of object band 422bp(hypB) is as shown in sequence in sequence table 12.
Three, high performance liquid chromatography detects for examination aspergillus flavus strain whether produce poison
Each is inoculated in to MEA slant tube substratum (formula: contain Fructus Hordei Germinatus in every liter of substratum and soak powder 30.0g, soy peptone 3.0g, agar 15.0g for examination aspergillus flavus strain; PH5.8) upper, cultivate 5 days for 28 ℃, add MEA slant tube substratum to rinse 5mL stroke-physiological saline solution, make bacteria suspension.Bacteria suspension is added in the test tube of 50mL, then add the nutrient solution (formula: solvent is water, and solute and concentration thereof are: sucrose 150g/L, yeast extract 20g/L, soy peptone 10g/L, pH5.9) of 10mL, cultivate 5 days for 30 ℃.By liquid fermentation liquid filter paper filtering, by 10 times of filtrate dilutions, get 10mL filtrate and join immune affinity column (Huaan, Beijing Mai Ke Bioisystech Co., Ltd product, its catalog number is HCM0125) in, after drain, with distilled water or deionized water wash 2 times, each 10mL, 2～3 drops/sec of flow velocitys; After drain, loading 1mL methyl alcohol, with sample bottle graft elutriant, after wash-out, liquid is for HPLC.High-efficient liquid phase chromatogram condition: C18 chromatographic column (4.6mm * 150mm, 5 μ m); Fluorimetric detector 2475 types, excitation wavelength 360nm, emission wavelength: 440nm; Column temperature: 30 ℃; Moving phase: take methyl alcohol: water (1:1, V:V) is moving phase; Sample size: 20 μ L; Flow velocity: 1.0mL/min.
Simultaneously with AFB
1, B
2, G
1, G
2hybrid standard product (LB96951) in contrast, are mixed with solution with methyl alcohol, according to as above condition, carry out high performance liquid chromatography detection.See whether take from each color atlas for the testing sample of examination aspergillus flavus strain has chromatographic peak to occur at the retention time place identical with aflatoxin standard substance.
Experiment in triplicate.
AFB
1, B
2, G
1, G
2the HPLC collection of illustrative plates of hybrid standard product (LB96951) as shown in Figure 2, as can be seen from the figure, AFG in aflatoxin standard substance
2retention time be 7.861min, AFG
1retention time be 8.793min, AFB
2retention time be 10.293min, AFB
1retention time be 11.735min.
In each strains tested, the HPLC collection of illustrative plates of Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12, DW-14, XinZ-11, XinZ-27, XinZ-33, YC-19, HG-14, QD-1, JZ-2 and GZ-17 does not all have chromatographic peak to produce at above four retention time places corresponding with aflatoxin standard substance.As can be seen here, these 10 bacterial strain aspergillus flavus not producing toxin.Wherein the HPLC collection of illustrative plates of Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12 as shown in Figure 3.
In each strains tested, the HPLC collection of illustrative plates of Aspergillus flavus (Aspergillus flavus) bacterial strain DW-5, DW-6, XinZ-15, XinZ-24, YC-10, HG-12, HG-24, QD-15, FX-1 and GZ-9 all has at least a chromatographic peak to produce at above four retention time places corresponding with aflatoxin standard substance.As can be seen here, these 10 bacterial strains produce aflatoxin.Wherein the HPLC collection of illustrative plates of Aspergillus flavus (Aspergillus flavus) bacterial strain DW-5 as shown in Figure 4.
For each, for examination aspergillus flavus strain, in the detected result of employing high performance liquid chromatography and step 2, adopt the qualification result of primer extension method provided by the invention in full accord.This has confirmed that primer extension method provided by the present invention identifies whether aspergillus flavus strain to be measured produces the accuracy of malicious method.
Comparative example 1, existing other pcr amplification methods identify whether aspergillus flavus strain to be measured produces malicious contrast
Strains tested: Aspergillus flavus (Aspergillus flavus) bacterial strain DW-12, DW-14, XinZ-11, XinZ-27, XinZ-33, YC-19, HG-14, QD-1, JZ-2, GZ-17.
Adopt patent application (denomination of invention: the primer sequence of authenticating aspergillus flavus not producing aspergillus flavus toxin by multiplex PCR and method; Application number: disclosed method 200910154898.7), take each genomic dna for examination Aspergillus flavus bacterial strain carries out pcr amplification as template.The target gene of pcr amplification is different from the present invention, is C3, aflT, norA and hypA.
Result shows, the C3 of strains tested DW-12, XinZ-11, XinZ-27 and QD-1 wherein, aflT, all there is (as shown in Figure 5) in a norA and hypA4 gene, according to patent (denomination of invention: the primer sequence of authenticating aspergillus flavus not producing aspergillus flavus toxin by multiplex PCR and method; Application number: the method for recording 200910154898.7), it may be toxigenic bacterium that these bacterial strains will be determined, but this four strains bacterium detects and is not toxigenic bacterium with HPLC in fact.Visible, by contrast, the accuracy of method provided by the present invention is higher.
Claims (10)
- For the identification of or the assistant identification aspergillus flavus strain to be measured primer pair group of whether producing aflatoxin, by following 4 primer pairs, formed: the primer pair 3 that in the primer pair 2 that in the primer pair 1 that in sequence table, two single stranded DNAs shown in sequence 1 and sequence 2 form, sequence table, two single stranded DNAs shown in sequence 3 and sequence 4 form, sequence table, two single stranded DNAs shown in sequence 5 and sequence 6 form, and the primer pair 4 that in sequence table, two single stranded DNAs shown in sequence 7 and sequence 8 form.
- 2. primer pair according to claim 1, is characterized in that: in described primer pair group, two primers of each primer pair are used at PCR reaction system moderate.
- 3. the test kit that contains primer pair group described in claim 1 or 2.
- 4. the preparation method of primer pair group described in claim 1 or 2, is characterized in that: described preparation method comprises the step that described two single stranded DNAs of each primer pair in primer pair group described in claim 1 or 2 are packed separately respectively.
- 5. the preparation method of test kit described in claim 3, comprise the steps: described two single stranded DNAs of each primer pair in the primer pair group described in claim 1 or 2 respectively separately after packing, be packaged in same reagent box with at least one in following substances: PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
- 6. primer pair group described in claim 1 or 2, or test kit claimed in claim 3 identifying or whether assistant identification aspergillus flavus strain to be measured produces the application in aflatoxin, or for the preparation of identifying or whether assistant identification aspergillus flavus strain to be measured produces the application in the product of aflatoxin.
- 7. utilize primer pair group described in claim 1 or 2, or the method that test kit claimed in claim 3 is identified or whether assistant identification aspergillus flavus strain to be measured produces aflatoxin, comprise the steps:(1) from aspergillus flavus strain to be measured, extract genomic dna as template, adopt 4 primer pairs in the primer pair group described in claim 1 or 2 to carry out respectively pcr amplification, obtain 4 parts of PCR products;(2) according to 4 parts of PCR products of step (1) gained, determine as follows whether aspergillus flavus strain to be measured produces aflatoxin: if the number of target DNA fragment is below 3 in described 4 parts of PCR products, described aspergillus flavus strain aspergillus flavus not producing toxin to be measured, or candidate's aspergillus flavus not producing toxin; If the number of target DNA fragment is 4 in described 4 parts of PCR products, described aspergillus flavus strain to be measured produces aflatoxin, or candidate produces aflatoxin;Described target DNA fragment is for arbitrary as follows: the DNA fragmentation that the size of utilizing described primer pair 1 amplification to obtain is 1141bp; The DNA fragmentation that the size of utilizing described primer pair 2 amplifications to obtain is 977bp; The DNA fragmentation that the size of utilizing described primer pair 3 amplifications to obtain is 629bp; The DNA fragmentation that the size of utilizing described primer pair 4 amplifications to obtain is 422bp.
- 8. method according to claim 7, is characterized in that: in step (1), while adopting described 4 primer pairs to carry out pcr amplification respectively, the annealing temperature of employing is 55 ℃.
- 9. according to the method described in claim 7 or 8, it is characterized in that: the nucleotides sequence of the DNA fragmentation that described size is 1141bp is classified as in sequence table shown in sequence 9; Described size is that the nucleotides sequence of the DNA fragmentation of 977bp is classified as in sequence table shown in sequence 10; Described size is that the nucleotides sequence of the DNA fragmentation of 629bp is classified as in sequence table shown in sequence 11; Described size is that the nucleotides sequence of the DNA fragmentation of 422bp is classified as in sequence table shown in sequence 12.
- 10. according to arbitrary described primer pair or test kit or method or application in claim 1-9, it is characterized in that: described aspergillus flavus strain to be measured for as lower any: Aspergillus flavus (Aspergillus flavus) DW-12, Aspergillus flavus (Aspergillus flavus) DW-14, Aspergillus flavus (Aspergillus flavus) XinZ-11, Aspergillus flavus (Aspergillus flavus) XinZ-27, Aspergillus flavus (Aspergillus flavus) XinZ-33, Aspergillus flavus (Aspergillus flavus) YC-19, Aspergillus flavus (Aspergillus flavus) HG-14, Aspergillus flavus (Aspergillus flavus) QD-1, Aspergillus flavus (Aspergillus flavus) JZ-2, Aspergillus flavus (Aspergillus flavus) GZ-17, Aspergillus flavus (Aspergillus flavus) DW-5, Aspergillus flavus (Aspergillus flavus) DW-6, Aspergillus flavus (Aspergillus flavus) XinZ-15, Aspergillus flavus (Aspergillus flavus) XinZ-24, Aspergillus flavus (Aspergillus flavus) YC-10, Aspergillus flavus (Aspergillus flavus) HG-12, Aspergillus flavus (Aspergillus flavus) HG-24, Aspergillus flavus (Aspergillus flavus) QD-15, Aspergillus flavus (Aspergillus flavus) FX-1 and Aspergillus flavus (Aspergillus flavus) GZ-9.
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| CN103937685A (en) * | 2014-04-23 | 2014-07-23 | 中国农业科学院农产品加工研究所 | Aspergillus flavus mixed strain incapable of producing aflatoxin and application thereof |
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| CN103937686A (en) * | 2014-04-23 | 2014-07-23 | 中国农业科学院农产品加工研究所 | Aspergillus flavus mixed strain incapable of producing aflatoxin and application thereof |
| CN103937687B (en) * | 2014-04-23 | 2016-04-13 | 中国农业科学院农产品加工研究所 | Do not produce flavus hybrid bacterial strain and the application thereof of aflatoxin |
| CN103937685B (en) * | 2014-04-23 | 2016-05-04 | 中国农业科学院农产品加工研究所 | A kind of aspergillus flavus hybrid bacterial strain and application thereof of aspergillus flavus not producing toxin |
| CN103937686B (en) * | 2014-04-23 | 2016-05-11 | 中国农业科学院农产品加工研究所 | A kind of aspergillus flavus Mixed Microbes and application thereof of aspergillus flavus not producing toxin |
| CN105219654A (en) * | 2015-09-25 | 2016-01-06 | 中国农业科学院油料作物研究所 | The aspergillus flavus strain of aflatoxin and the application in aflatoxin pollution of peanuts biological control thereof are not produced in one strain |
| CN105219654B (en) * | 2015-09-25 | 2018-09-04 | 中国农业科学院油料作物研究所 | One plant of aspergillus flavus strain for not producing aflatoxin and its application in aflatoxin pollution of peanuts biological control |
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