CN109468367A - Aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount and its detection method - Google Patents
Aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount and its detection method Download PDFInfo
- Publication number
- CN109468367A CN109468367A CN201811494322.0A CN201811494322A CN109468367A CN 109468367 A CN109468367 A CN 109468367A CN 201811494322 A CN201811494322 A CN 201811494322A CN 109468367 A CN109468367 A CN 109468367A
- Authority
- CN
- China
- Prior art keywords
- aflatoxin
- pcr
- genetic transcription
- probe
- transcription amount
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to biological fields, and in particular to a kind of aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount and its detection method.The present invention establishes synchronous detection aspergillus flavus strain and produces poison amount and the synchronous RT-PCR detection method of nor-1 genetic transcription amount, gained aspergillus flavus strain, which is detected, using the method produces poison amount, nor-1 genetic transcription amount quantifies aflatoxin with using HPLC, Nanodrop quantifies the result of Nor-1 genetic transcription amount, and there are good linear relationships, therefore, the ratio result of AFT/Nor-1 based on synchronous detection RT-PCR method acquisition is reliable, accurately, it can be used as the identification of indicator for determining that aspergillus flavus strain produces virulence height, it realizes and the quick of virulence is produced to aspergillus flavus strain, accurate characterization and evaluation, it is of great significance to aflatoxin contamination early warning with prevention and control.
Description
Technical field
The invention belongs to biological fields, and in particular to a kind of aflatoxin yield is synchronous with Nor-1 genetic transcription amount
Detect RT-PCR kit and its detection method.
Background technique
Aflatoxin is to find the strongest a kind of mycotoxin of toxicity, by taking aflatoxin B1 as an example, toxicity so far
It is 10 times of potassium cyanide, 68 times of arsenic, I class carcinogenic substance is classified as by international cancer tissue.Aflatoxin easily pollute peanut,
The grain and oil crops product such as corn, rice, also numerous plant products such as pollution walnut, American pistachios and Chinese medicine.Occur both at home and abroad
Cross the people and animals group poisoning that a lot of aflatoxin cause.According to International Agency for Research on Cancer (IARC) latest report, the whole world is only
Developing country just has about 500,000,000 populations to endure aflatoxin exposure to the fullest extent.China is the heavier area of aflatoxin contamination.Agriculture
Industry portion many years findings of the survey show China's staple crops product by aflatoxin contamination in persistently aggravating trend, seriously
Area's content of toxins is more than hundreds times of limit standard；Although strong toxigenic bacterium strain accounting has become threat farming less than 20%
The major hidden danger of object Product quality and safety.
Aflatoxin is mainly generated by fungies such as aspergillus flavus, aspergillus parasiticus.Existing research shows different aspergillus flavus strains
Generating the ability of aflatoxin --- i.e. production virulence can differ hundreds times, and strong toxigenic bacterium strain is to cause the important source of high pollution
Head, but lack the effective ways that specificity quickly identifies strong toxigenic bacterium strain so far.Identify the side that aspergillus flavus strain produces virulence at present
There are two main classes for method: one kind is to cultivate bacterial strain directly to evaluate it by measuring its amount for generating aflatoxin after a certain period of time
Toxin producing C.Such methods first is that the time it is long, it is necessary to isolate bacterial strain first, be further cultured for, finally by detection aflatoxin
Content is evaluated；Second is that the impacted factor of the biosynthesis of aflatoxin is very more and complicated, same strain culturing is different
Aflatoxin volume variance between culture batch is big, is as a result difficult to be used as itself production poison spy that characterization bacterial strain should be constant
Property, to influence the accurate reliability of this method Identification Evaluation result.Another kind of method is to produce malicious related gene by measurement to turn
Record level produces virulence to evaluate bacterial strain, has document report detection Nor-1 gene evaluation to produce virulence.But the deficiency of such methods
Place is: just have at least about 20% bacterial strain under natural conditions causes because detecting the missing of the malicious related gene of production other than gene
Poison is not produced naturally, but detect gene can still express, so as to cause the spurious results of such methods.
Summary of the invention
The present invention is in view of the deficiencies of the prior art, and it is an object of the present invention to provide a kind of aflatoxin yield and Nor-1 gene turn
The synchronous detection RT-PCR kit and its detection method of record amount.
For achieving the above object, the technical solution adopted by the present invention are as follows:
A kind of aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount, comprising:
1) it is used to expand drawing for bacteriophage institute's released dna segment of surface display aflatoxin antiidiotype nano antibody
Object and probe: upstream primer Ph-F, nucleotide sequence is as shown in SEQ ID NO.1；Downstream primer Ph-R, nucleotide sequence is such as
Shown in SEQ ID NO.2；Fluorescence probe Ph-probe, nucleotide sequence is as shown in SEQ ID NO.3；
2) for expanding the primer and probe of Tq-nor-1 gene: upstream primer Tq-nor1-F, nucleotide sequence such as SEQ
Shown in ID NO.4；Downstream primer Tq-nor1-R, nucleotide sequence is as shown in SEQ ID NO.5；Fluorescence probe Tq-probe, core
Nucleotide sequence is as shown in SEQ ID NO.6；
3) universal probe qPCR premixed liquid, archaeal dna polymerase, MgCl2、dNTPs、H2O。
According to the above scheme, the upstream and downstream primer: Ph-F, Ph-R, Tq-nor1-F, Tq-nor1-R are anti-in PT-PCR amplification
Answering the final concentration in system is 300~400nM.
According to the above scheme, the fluorescence probe: end of the Ph-probe and Tq-probe in PT-PCR amplification reaction system
Concentration is 200~400nM.
According to the above scheme, the archaeal dna polymerase final dosage in PT-PCR amplification reaction system is 0.5U~1.0U, institute
State MgCl2Final concentration of 1mM~2mM in PT-PCR amplification reaction system, the dNTPs is in PT-PCR amplification reaction system
In final concentration of 200uM~400uM.
1) and 2) according to the above scheme, fluorescence probe both ends described in are connected separately with fluorophor and quenching group, described
Fluorophor is selected from FAM, TET, VIC, HEX, and the quenching group is selected from TAMRA, BHQ1, BHQ2, BHQ3 or other quenchers.
Using mentioned reagent box with the method for one step RT-PCR detection aflatoxin yield and Nor-1 genetic transcription amount, packet
Include following steps:
(1) foundation of quantitative aflatoxin S type standard curve: the Immune competition stage of reaction, in aflatoxin Dan Ke
It is anti-only using the aflatoxin mark product and surface display aflatoxin of various concentration in the case that grand antibody package amount is certain
The bacteriophage competitive binding aflatoxin monoclonal antibody of special type nano antibody, Immune competition after reaction, are incorporated into
The bacteriophage elution of surface display aflatoxin antiidiotype nano antibody in aflatoxin monoclonal antibody is different dense
The corresponding bacteriophage for eluting different amounts of surface display aflatoxin antiidiotype nano antibody of the aflatoxin mark product of degree,
Bacteriophage in eluent understands released dna molecule during PCR is heated, and the DNA molecular of release is as in RT-PCR reaction
Amplification target, each eluent is synchronized by RT-PCR amplified reaction using the kit respectively, after amplified reaction
Different Ct values is obtained, using the logarithm of aflatoxin concentration as abscissa, using Ct value as ordinate, carries out regression analysis,
Obtain the quantitative S type standard curve of aflatoxin；
(2) foundation of Nor-1 genetic transcription amount RT-PCR standard curve: will be known to nor-1 gene DNA fragment Tq-nor1
Then the sample serial dilution of copy number carries out each copy number Tq-nor1 using the kit respectively at different copy numbers
With one step RT-PCR amplified reaction, different Ct values is obtained after amplified reaction, with the logarithm of Tq-nor1 copy number for horizontal seat
Mark carries out regression analysis, obtains the quantitation curves of nor-1 genetic transcription amount using Ct value as ordinate；
(3) aspergillus flavus strain is cultivated, Aspergillus flavus spore is taken to carry out shaken cultivation, after culture, filters to take bacterial strain training
Nutrient solution and Aspergillus flavus pompon；After strain cultured solution is diluted certain multiple, the Huang in alternative steps (1) described immune response is bent
Mould toxin mark product participate in Immune competition reaction, and the surface in aflatoxin monoclonal antibody is incorporated into after Immune competition reaction
Show aflatoxin antiidiotype nano antibody bacteriophage elution, the DNA molecular that eluent pnagus medius is discharged as
With the template of quantitative amplification aflatoxin in one step RT-PCR amplified reaction；Separately by after the drying of Aspergillus flavus pompon, total serum IgE is extracted
And reverse transcription is used as after the cDNA is diluted certain multiple with quantitative amplification Nor-1 base in one step RT-PCR amplified reaction at cDNA
The template of cause；
(4) RT-PCR amplified reaction is synchronized using the DNA molecular of bacteriophage release and the cDNA as template, expanded
Increase and obtain two Ct values after reaction, two Ct values are substituted into the quantitative S type standard curve and nor-1 of aflatoxin respectively
The quantitation curves of genetic transcription amount, conversion obtain aflatoxin concentration and nor-1 genetic transcription amount, determine yellow song with this
The ratio of mould Output of toxin and Nor-1 genetic transcription amount.
According to the above scheme, the bacteriophage of the surface display aflatoxin antiidiotype nano antibody is bacteriophage
VHH 2-5, the aflatoxin monoclonal antibody are aflatoxin monoclonal antibody 1C11.
According to the above scheme, in the reaction system of the same one step RT-PCR amplified reaction, upstream and downstream primer: Ph-F, Ph-R,
The final concentration of Tq-nor1-F, Tq-nor1-R are 300~400nM；Fluorescence probe: the final concentration of Ph-probe and Tq-probe
200~400nM；The final dosage of archaeal dna polymerase: 0.5U~1.0U；MgCl2Final concentration: 1mM~2mM；DNTPs final concentration:
200uM~400uM.
According to the above scheme, the reaction system of the same one step RT-PCR amplified reaction includes: universal probe qPCR premixed liquid 5
μ L, 0.1 Ph-F μ L, 0.1 Ph-R μ L, 0.1 Ph-probe μ L, 2 μ L of bacteriophage template, 0.1 Tq-nor1-F μ L, Tq-
0.1 μ L of nor1-R, 0.1 Tq-probe μ L, 1 μ L of Nor-1 gene template, 0.2 μ L of archaeal dna polymerase, MgCl2 0.8μL、dNTPs
0.2 μ L, adds H210 μ L of O polishing.
According to the above scheme, the amplification condition of the same one step RT-PCR amplified reaction is 95 DEG C, 5min；95 DEG C, 10s, 60 DEG C
30s, 40 circulations.
According to the above scheme, aflatoxin concentration model in the quantitative S type standard curve of step (1) described aflatoxin
Enclosing for 33.33ng/mL~1.69pg/mL, the lowest detection line LOD of aflatoxin is 0.018ng/mL；Step (2) is described
Nor-1 gene copy number range is 10 in the quantitation curves of nor-1 genetic transcription amount2~108。
According to the above scheme, culture medium used in the culture aspergillus flavus strain be CDA culture medium, condition of culture: 28 DEG C,
It is cultivated 10 days under 90% humidity；Culture medium used in the spore shaken cultivation for taking Aspergillus flavus is potato glucose liquid
Culture medium；Condition of culture: at 28 DEG C, shaken cultivation 96h under the conditions of 200rpm.
Beneficial effects of the present invention are as follows:
(1) present invention establishes aspergillus flavus strain and produces poison amount and the synchronous RT-PCR detection method of nor-1 genetic transcription amount,
Reagent dosage is less in the method, and cost is lower, and high-throughput detection may be implemented；And with one step RT-PCR quantitative detection aspergillus flavus
Bacterial strain produces poison amount and the result of nor-1 genetic transcription amount is reliable, and method is stablized, reproducible；
(2) poison amount, nor-1 genetic transcription are produced using aspergillus flavus strain obtained by synchronous detection RT-PCR method of the present invention
Amount with aflatoxin is quantified using HPLC, Nanodrop quantifies the result of Nor-1 genetic transcription amount there are good linear passes
System, therefore, (aspergillus flavus strain produces malicious amount/nor-1 genetic transcription to the AFT/Nor-1 obtained based on synchronous detection RT-PCR method
Amount) ratio result it is reliable, accurate, can be used as the identification of indicator for determining that aspergillus flavus strain produces virulence height, realize to Huang
Aspergillus strain produces the fast and accurately characterization and evaluation of virulence, is of great significance to aflatoxin contamination early warning with prevention and control；
(3) synchronous detection RT-PCR method of the present invention has simplified analytical model, optimizes experimentation and structure, and
Synchronization Analysis for other small-molecule substances in aflatoxin and its synthesis access provides detection platform and theoretical basis.
Detailed description of the invention
Fig. 1 is archaeal dna polymerase, dNTPs and MgCl in optimized synchronization RT-PCR reaction2Concentration.
Fig. 2 is to assess with one step RT-PCR amplification efficiency.
Fig. 3 is the quantitation curves that aflatoxin B1 and nor-1 genetic transcription amount are quantified with one step RT-PCR.
Fig. 4 is quantitative with one step RT-PCR: with aflatoxin B1, B2, G1, G2, ZEN, DON, FB1 cross reacting rate.
Fig. 5 is to compare with one step RT-PCR and HPLC and Nanodrop quantitative result.
Fig. 6 is aflatoxin yield and Nor-1 gene expression amount correlativity (A), aflatoxin yield and AFT/
Nor-1 ratio correlativity (B).
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1
One, research has verified aflatoxin yield and Nor-1 genetic transcription amount ratio with extraordinary relatively stable
Property
Weigh 1g NaNO3、1g K2HPO4、0.5g MgSO4·7H2O、0.5g KCl、0.01g FeSO4, 30g glucose
With 20g agar powder, being settled to total volume with deionized water is 1000ml, 121 DEG C of high-temp steam sterilizing 30min, is prepared into CDA training
Support base.With CDA solid culture based on being cultivated aspergillus flavus strain 10 days under 28 DEG C, 90% humidity, then washed with 20% Tween-20
Culture plate is to obtain Aspergillus flavus spores solution.Using blood counting chamber counting method, by Aspergillus flavus spores solution vortex
Concussion instrument concussion is uniform, with microscope to Aspergillus flavus spores solution microscopic count.
15mL potato dextrose broth is placed in 50mL triangular flask, 121 DEG C of high pressure sterilization 30min, according to meter
Number is as a result, be added Aspergillus flavus spores solution to final concentration of every milliliter 1 × 105A spore shakes under the conditions of 200rpm at 28 DEG C
Swing culture 96h.With double-layer filter paper broth filtrate, filtrate (saving backup) and Aspergillus flavus mycelium pellet is obtained by filtration.For Huang
Aspergillus hyphae ball squeezes excessive moisture with filter paper, dries 12h under the conditions of 65 DEG C with baking oven, obtains dry mycelium, be cooled to
Room temperature saves backup under the conditions of being placed on -70 DEG C, saves backup under the conditions of 4 DEG C of the filtrate being obtained by filtration.
Using affine in immunity purification-high performance liquid chromatography standard method, obtained after measuring Aspergillus flavus spore shaken cultivation
Filtrate (above-mentioned to save backup) in aflatoxin concentration, using conventional Nor-1 genetic transcription with respect to quantity measuring method,
Measure Nor-1 genetic transcription relative quantity in the above-mentioned dry mycelium saved backup.
Using same operation as described above, 7 aspergillus flavus strains are at different time sections culture assay two batches one after another
It is secondary as a result, measurement result is shown in Table 1.
1 aspergillus flavus strain of table produces aflatoxin and Nor-1 genetic transcription relative quantity measurement result
According to 1 measurement result of table, two batch aflatoxin concentration differences are larger, therefore only according to aflatoxin yield
It is not able to satisfy evaluation aspergillus flavus strain and produces virulence；When some aspergillus flavus strain Nor-1 genetic transcription relative quantities is high, there is production poison unexpectedly
Power relatively low situation instead；In addition toxigenic bacterium strain can not transcribe Nor-1 gene yet, therefore can not also expire only according to Nor-1 gene
Foot evaluation aspergillus flavus strain produces virulence.It is surprising that turning in table 1 when aflatoxin concentration value and Nor-1 gene
Ratio --- [AFT]/[nor-1] numerical value i.e. in table 1 that record relative magnitude obtains after being divided by, discovery present very strong rule
Rule property, 7 bacterial strains production virulence sequence consensus that not only culture assay batch obtains twice, but also [AFT] of each bacterial strain/
[nor-1] numerical value has relative stability, produces virulence evaluation so as to meet aspergillus flavus strain.
If from 1 data of table as can be seen that using aflatoxin concentration value and Nor-1 genetic transcription relative magnitude
Ratio produces virulence to evaluate aspergillus flavus strain, accuracy and reliability be substantially better than individually using aflatoxin concentration value or
Individually use and Nor-1 genetic transcription relative magnitude method.
The foundation of 2 aflatoxin yield of embodiment detection RT-PCR method synchronous with Nor-1 genetic transcription amount
Utilize bacteriophage VHH 2-5 and the nor-1 base of existing surface display aflatoxin antiidiotype nano antibody
Because of DNA fragmentation Tq-nor1, aflatoxin yield detection RT-PCR method synchronous with Nor-1 genetic transcription amount is established, according to
Above-mentioned testing result produces the scientific basis of virulence as characterization and evaluation aspergillus flavus strain, evaluates aspergillus flavus strain for Rapid identification and produces
Virulence provides method support.Specific step is as follows for these.
The bacteriophage VHH 2-5 of the surface display aflatoxin antiidiotype nano antibody is the Chinese Academy of Agricultural Sciences
Oil crops research institute quality testing center is developed, in periodical literature " Yanru Wang；Peiwu Li；Zuzana
Majkova；Candace R.S.Bever；Hee Joo Kim；Qi Zhang；Julie E.Dechant；Shirley J.Gee；
Bruce D.Hammock；Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain
Antibody for the Aflatoxin Immunoassay；Analytical Chemistry, 2013,8298-8303 " report
Road mistake, bacteriophage used in the present embodiment is that laboratory is saved previously in Escherichia coli ER2738, passes through amplification and obtains.Expand
Increasing method is as follows:
Random picking monoclonal, is inoculated into from the ER2738 single colonie plate for possessing nano antibody bacteriophage VHH 2-5
Containing in 1mL SB- ammonia benzyl fluid nutrient medium, the 225rpm overnight incubation in 37 DEG C of constant-temperature tables；Bacterium solution is incubated overnight by above-mentioned
It is added in 100mL SB- ammonia benzyl fluid nutrient medium, 225rpm, 37 DEG C of cultures to OD600=0.5-0.6；1.5ml is added
(titre is 1 × 10 for M13KO7 helper phage11-1×1012Pfu/mL) into cultured bacterium solution, 37 DEG C of standing 30min；?
The card that final concentration of 70 μ g/mL is added in bacterium solution receives mycin, and 37 DEG C, 225rpm, shaken cultivation is overnight；4 DEG C, 10000rpm centrifugation
The bacterium solution 15min being incubated overnight, takes supernatant, and is transferred in clean centrifugal bottle；The PEG/NaCl of 1/4 volume, Yu Bing is added
Upper standing 2h；4 DEG C, 10000rpm be centrifuged 30min, will precipitating with 2mL 0.5%BSA/PBS be resuspended, then in 12000rpm from
Heart 5min, takes supernatant, and supernatant is filtered with 0.22 μm of filter and is mixed with isometric sterile glycerol, is dispensed, titre is surveyed.Titre
Measuring method is as follows:
Random picking monoclonal, is inoculated into the LB liquid containing 0.04mg/mL tetracycline from ER2738 single colonie plate
In culture medium, the 225rpm overnight incubation in 37 DEG C of constant-temperature tables；The above-mentioned 20 μ L of bacterium solution that is incubated overnight is taken to add to 2mL SB culture
Base, 225rpm, 37 DEG C of cultures to OD600≈1；Gradient dilution is carried out to the bacteriophage for needing to measure titre with LB liquid medium；
Each dilution takes 10 μ L respectively, is added to 100 μ L OD600In the ER2738 bacterium solution of ≈ 1,37 DEG C of standing 20min make bacteriophage
Infect Escherichia coli；Escherichia coli bacteria liquid after infecting is coated on LB- ammonia benzyl plate, is just set in 37 DEG C of constant incubators
Then 30min is inverted overnight incubation；Carry out plate count of the single colonie quantity at 100 or so is chosen, is come according to following formula
Calculate the titre of bacteriophage:
The preparation method of the nor-1 gene DNA fragment Tq-nor1: 15mL potato dextrose broth is set
In 50mL triangular flask, the spore liquid of N73 Aspergillus flavus is added to final concentration of 1 × 10 in 121 DEG C of high pressure sterilization 30min5ml-1,
At 28 DEG C, under the conditions of 200rpm after shaken cultivation 96h；Double-layer filter paper squeezes mycelium pellet culture solution, 65 DEG C of dry 12h, liquid nitrogen
It is quick-frozen, it pulverizes；Precise 200mg mycelia powder is pressed with DNA extraction kit (DNeasy Plant Mini Kit)
The genomic DNA that N73 bacterial strain is extracted according to kit specification, goes out the nor-1 gene that size is 400bp with following primer amplification
Fragment products, and with E.Z.N.A.TM (OMEGA) gel extraction kit, the DNA piece that purifying size is 400bp to specifications
Section, can be obtained Tq-nor1.
Nor1-F:5'-ACCGCTACGCCGGCACTCTCGGCAC-3'；
Nor1-R:5'-GTTGGCCGCCAGCTTCGACACTCCG-3'。
One, aflatoxin yield detection RT-PCR method synchronous with Nor-1 genetic transcription amount is established
1, the primer and probe sequence design of aflatoxin yield detection RT-PCR synchronous with Nor-1 genetic transcription amount
The DNA fragmentation and nor- discharged with the bacteriophage VHH2-5 of surface display aflatoxin antiidiotype nano antibody
The DNA fragmentation Tq-nor1 of 1 gene is that template carries out RT-PCR synchronous amplification, is determined by the amplification of same one step RT-PCR yellow
The ratio of aspertoxin yield and Nor-1 genetic transcription amount.
According to coding nano antibody in the bacteriophage VHH 2-5 of surface display aflatoxin antiidiotype nano antibody
Sequence and nor-1 gene order, the upstream and downstream primer (Ph-F, Ph-R) and probe (Ph-probe) sequence, Tq- of aflatoxin
The upstream and downstream primer (Tq-nor1-F, Tq-nor1-R) and probe (Tq-probe) sequence of nor-1, then according to dual RT-PCR
Primer and probe design principle and points for attention, are verified with Oligo7.0 Primer Analysis Software in reaction.
Primer and probe verify principle: the TM value of all primers should set identical or close level, and should make as far as possible
The TM value of all probes approaches and is greater than about 5~10 DEG C of primer TM value；Due to being the synchronous reaction of same system, it is ensured that Suo Youyin
Object and probe are not easy to form dimer；Blast search, it is ensured that primer and probe has specificity to purpose target spot.
Following primer and probe sequence determination meet the above principles and requirements, summarize such as table 2 shown in.
2 Multiplex real-time PCR primer and probe sequence of table
2, the response parameter of aflatoxin yield RT-PCR detection method synchronous with Nor-1 genetic transcription amount
With one step RT-PCR response parameter be different from term single gene amplification RT-PCR because to fully consider reaction system it
Between interfere with each other.Firstly, by two kinds of substances --- aflatoxin antiidiotype nano antibody bacteriophage specific DNA fragment and
The RT-PCR of nor-1 gene DNA fragment is expanded, and reactive component, which directly mixes, does not add other components, and it is anti-to carry out dual RT-PCR
It answers, as a result as shown in Figure 1A, Cong Tuzhong is we have seen that the amplification of VHH 2-5 phage DNA molecule is significantly inhibited.It is dual
In RT-PCR reaction, the amplification efficiency and target sequence of different amplified matters all may be different, the low sample of amplification efficiency or low rich
The amplification for spending target sequence may be inhibited by the target sequence of high amplification sample or more high abundance.
For this purpose, the present invention reacts aflatoxin yield with the RT-PCR of Nor-1 genetic transcription amount synchronization detecting method
Condition optimizes.Archaeal dna polymerase, MgCl are optimized in the present invention2With the concentration of dNTPs.Shown in Figure 1B the result shows that,
VHH2-5 phagocytosis bulk concentration is 106It is additional to add dNTPs and MgCl when pfu/mL2Afterwards, cycle threshold Ct moves forward, in relatively early circulation
There is amplification curve when number, explanation improves the amplification of VHH 2-5 phage DNA molecule as a result,.Fig. 1 C is shown when DNA polymerize
When enzyme increases to 1.0U from 0.25U, the amplification efficiency of bacteriophage is also significantly improved.Therefore, more early go out according to threshold cycle Ct
Now with amplification curve in exponential phase closer to the principle of S type, archaeal dna polymerase of the present invention, MgCl2With the preferable amount model of dNTPs
It encloses are as follows:
Archaeal dna polymerase: 0.5U~1.0U；MgCl2: 1mM~2mM；DNTPs:200uM~400uM.
The present invention according to the above results, finally gives the amplified reaction parameter of optimization, is specifically shown in Table 3.
The RT-PCR response parameter of 3 aflatoxin of table and Nor-1 genetic transcription amount synchronization detecting method
3, the amplification efficiency of aflatoxin yield detection RT-PCR method synchronous with Nor-1 genetic transcription amount
The same one step RT-PCR amplification of the bacteriophage being serially diluted and the nor-1 being serially diluted are shown in same one step RT-PCR amplification
Curve (RFU, Relative fluorescence units), as shown in Figure 2 A.Fig. 2 B is the amplification efficiency standard curve obtained according to amplification curve, is bitten
The slope of standard curve -3.37 of thallus VHH 2-5 amplification efficiency, according to the calculation formula (E=[10 of amplification efficiency E1/-slope-1]
× 100%) it calculates, when amplification efficiency E is 98.03%, the detectable concentration range of VHH 2-5 bacteriophage is 109~
103pfu/mL.When the copy number of nor-1 gene DNA fragment Tq-nor1 is 102-108When copy, nor-1 gene magnification is similarly obtained
Efficiency is 90.25%.Amplification efficiency E is all satisfied the area requirement of 90%-105%, and amplification efficiency calibration curve coefficient correlation
R2>0.99.Therefore, the same one step RT-PCR system of optimization can be used for VHH 2-5 bacteriophage and the synchronous high-efficiency of nor-1 gene expands
Increase.
Two, quantitation curves are established and synchronous RT-PCR method is assessed
1. the S type standard curve of quantitative aflatoxin
1.1 immune response
(1) be coated with: with PBS by commercially available aflatoxin monoclonal antibody 1C11 (deposit number be CCTCC NO:
The hybridoma cell strain 1C11 of C201013, which secretes, to be generated, and number of patent application is that CN201010245095.5 has specific report)) it is dilute
It releases to 1.0 μ g/mL, is added in 96-well ELISA Plate with micropipettor, every 100 μ L of hole, in 4 DEG C of overnight incubation (≈
12h), PBST board-washing 3 times；Closing: being closed, every 300 μ L of hole with confining liquid, in 37 DEG C of incubation 45min, PBST board-washing 3 times；
(2) it closes: being closed with confining liquid, every 300 μ L of hole, in 37 DEG C of incubation 45min, PBST board-washing 3 times；
(3) it competes: by aflatoxin B1 mark product, being 200ng/mL with 100% pure methanol dilution to concentration, then use
10% 3 times of (v/v) methanol/PBS gradient doubling dilution aflatoxin B1 standard solution, makes concentration range 33.33ng/mL-
1.69pg/mL；Then by the VHH 2-5 bacteriophage (1.0 × 10 of 50 μ L known concentrations10Cfu/mL) with the Huang of 50 μ L series of concentrations
100 μ L mixtures are added to 96-well enzyme mark version micropore and are washed after 37 DEG C of incubation 1h with PBST by aspertoxin B1 mixing
ELISA Plate 10 times；
(4) elute: 90 μ L bacteriophage elution liquid are injected in every hole, and 37 DEG C of standing warm bath 15min are gently blown with micropipettor
It packs and is transferred out of the eluent containing phasmid；
(5) neutralize: 90 μ L, which remove liquid and the mixing of 10 μ L neutralizers, makes mixed liquor at neutrality --- the volume of neutralizer is added
It is adjusted according to the actual pH of eluent and neutralizer, guarantees mixed liquor at neutrality；
1.2 establish standard curve: immune response stage, the certain situation of aflatoxin monoclonal antibody 1C11 package amount
Under, the aflatoxin of various concentration can be got over different amounts of VHH 2-5 bacteriophage competitive binding 1C11, aflatoxin concentration
Greatly, for bacteriophage in conjunction with the chance of 1C11 with regard to smaller, binding capacity is lower；After immune response, the phagocytosis that is incorporated on 1C11
Body elutes, and the bacteriophage quantity in eluent is related to aflatoxin concentration, and the phasmid in eluent is reacted in PCR to be heated
Process can released dna molecule, the DNA molecular of release is as the amplification target in RT-PCR reaction, and fluorescence is determined after amplified reaction
Amount system software can provide the Ct value of the Phage amplification of different number in the corresponding different eluents of not same amount aflatoxin,
The Ct value that the aflatoxin (33.33ng/mL~1.69pg/mL) for the various concentration being serially diluted obtains is dense to aflatoxin
The logarithm of degree carries out four parameter logistic regressions with 8.0 software of Origin Pro, the S type standard as quantitative aflatoxin
Curve, as shown in Figure 3A.
2.nor-1 the quantitation curves of genetic transcription amount are established
Aspergillus flavus strain is taken, the present embodiment uses the N73 aspergillus flavus strain of this research center preservation, is high toxigenic bacterium
Strain, for Tq-nor1 preparation in this research.Other aspergillus flavus strains can use " the nor-1 gene DNA fragment Tq- being described above
The preparation method of nor1 " amplifies the DNA fragmentation Tq-nor1 that size is 400bp, can be used as and establishes nor-1 genetic transcription amount
Quantitation curves alternative bacterial strain.Spectrophotometer (NanoDrop 2000, Thermo Scientific, U.S.A.) inspection
After surveying Tq-nor1 concentration, the copy number of Tq-nor1 is calculated.Tq-nor1 carries out serial dilution (10 as known quantity sample2
~108Copies after), RT-PCR amplification is carried out, with 8.0 software of Origin Pro with Tq-nor1 standard sample series copy number
Logarithm is abscissa, and Ct value is ordinate, carries out regression analysis, and the quantitative criterion if Fig. 3 B is nor-1 genetic transcription amount is bent
Line.
The quantitation curves of aflatoxin are as shown in Figure 3A.As seen from the figure, aflatoxin is quantified with one step RT-PCR
The detection limit LOD of B1 (uses IC10Indicate) it is 0.018ng/mL, therefore the RT-PCR quantitative detection aflatoxin B1 established has
Very high sensitivity.In addition, Fig. 3 B is disclosed, can quantify nor-1 gene copy number range with one step RT-PCR is 102~108, this
Sufficiently confirm that the same one step RT-PCR established has apparent advantage in terms of low-level nor-1 gene absolute quantitation.
3. with the cross reacting rate measurement of one step RT-PCR detection aflatoxin
Aflatoxin B 2, G1, G2, zearalenone (ZEN), deoxynivalenol (DON), fumonisin
(FB1) standard items are diluted to a series of concentration, after competitive immunoreaction occurs with bacteriophage VHH 2-5, by ELISA Plate hole bear building-up
It closes the bacteriophage elution on monoclonal antibody 1C11 to come out with Tq-nor1 progress RT-PCR synchronous amplification, the Ct expanded
Value corresponds to toxin various concentration logarithm and does standard curve, calculates IC50Value, according to cross reaction calculation formula %CR=
(IC50AFB1/IC50analyte) × 100 calculate cross reacting rate.As shown in figure 4, being directed to the friendship of aflatoxin B1, B2, G1, G2
Pitching reactivity is respectively 100%, 101%, 34% and 12%.It can be seen that this method can be realized to total aflatoxin content
Measurement.Wherein, the cross reacting rate of aflatoxin G 1 and G2 are respectively 34% and 12%, but do not influence this method and identifying
Aspergillus flavus produces the application in terms of virulence, because aspergillus flavus strain only generates B same clan aflatoxin.The intersection of aflatoxin B 2
Reactivity is 101%, and the RT-PCR method of foundation quantifies aflatoxin B 2 with higher sensitivity, this is to a certain extent
The RT-PCR method identification aspergillus flavus for improving foundation produces the reliability of virulence, because aspergillus flavus strain can produce aspergillus flavus poison
Plain B1 also can produce aflatoxin B 2.Thus, it is to B1 and B2 with the production virulence of the RT-PCR identification aspergillus flavus strain of foundation
The evaluation of comprehensive yield.
4. adding recycling verifying with one step RT-PCR
Aflatoxin B1 mark product and nor-1 gene DNA fragment Tq-nor-1 are added simultaneously in 2mL blank PDB culture medium.
Concussion mixes, and mixed liquor is placed in 4 DEG C of avoid light places 4 days after mixing.After 4 days, 20 times of mixed liquor are diluted, RT-PCR is used
The content of aflatoxin B1 and Tq-nor-1 in synchronous detection mixed liquor.3 repetitions are set in group on the same day, not same date
3 repetitions are set between group.Addition recycling the results are shown in Table 3:
The measurement of 3 TIANZHU XINGNAO Capsul of table
The TIANZHU XINGNAO Capsul of aflatoxin B1 is 88.37%~103.10%, Nor-1 gene DNA fragment Tq-nor-1
TIANZHU XINGNAO Capsul be 86.18%~98.17%, this result shows that, the same one step RT-PCR of foundation is tested and analyzed in actual sample
In, there is reliable repetition and reproducibility.
5. production poison amount and nor-1 gene expression dose that the synchronous RT-PCR method of application quantifies aflatoxin bacterial strain
This research has chosen 17 plants with the different aspergillus flavus strains for producing virulence.By 15mL potato glucose Liquid Culture
Base is placed in 50mL triangular flask, and aspergillus spore liquid is added to final concentration of 1 × 10 in 121 DEG C of high pressure sterilization 30min5ml-1.28
DEG C, under the conditions of 200rpm after shaken cultivation 96h, with double-layer filter paper broth filtrate, strain cultured solution and aspergillus flavus is obtained by filtration
Mycelium pellet, Aspergillus flavus pompon squeeze excessive moisture with filter paper, dry 12h under the conditions of 65 DEG C with baking oven, are cooled to room temperature
Afterwards, with liquid nitrogen grinding at powder, each sample precise 0.20mg mycelia powder.According to RNA extracts kit (RNeasy
Plant Mini Kit) specification progress Total RNAs extraction.Then cDNA is synthesized with QuantiTect reverse transcription reagent box.It should
Replace after 100~1000 times of cDNA solution dilutions with the Tq-nor1 in one step RT-PCR amplification system, as one of amplification template into
Row is expanded with one step RT-PCR, measures nor-1 genetic transcription amount.After strain cultured solution dilutes 10 times with 10% (w/v) BSA/PBS,
Immune competition reaction is participated in instead of the aflatoxin mark product in immune response, the phasmid conduct after competitive reaction in eluent
With another template of one step RT-PCR amplification system, synchronizes RT-PCR amplification assay bacterial strain and produce poison amount.With same one step RT-PCR side
The production poison amount and nor-1 gene expression dose of 17 plants of Aspergillus flavus of standard measure, as a result such as table 4.
Table 4 quantifies the production poison amount and nor-1 gene expression dose result of 17 plants of Aspergillus flavus with one step RT-PCR
It is yellow bent with HPLC standard measure with reference to standard GB/T 5009.22-2016 method for the accuracy of verification result
Trichoderma strain produces aflatoxin amount；Meanwhile bacterial strain nor-1 gene expression amount is quantified with spectrophotometer (Nanodrop), it is quantitative
As a result such as table 4.And result is compared with the result that same one step RT-PCR obtains, comparison result such as Fig. 5.Fig. 5-A is RT-PCR
Aflatoxin comparison result is quantified with HPLC, obtained equation of linear regression is Y=0.947X -3.84, linear regression analysis
Produce good correlation (R2=0.999).Fig. 5-B quantifies Nor-1 genetic transcription amount for RT-PCR and Nanodrop and compares
As a result, the equation of linear regression that method obtains is Y=1.05X -1.18, coefficient R2=0.989.Different detection methods compare
As a result illustrate that the same one step RT-PCR quantitative result established is reliable, can be used for aspergillus flavus strain and produce poison amount and nor-1 genetic transcription amount
Synchronization Analysis.
Embodiment 3
One, aspergillus flavus is evaluated using Nor-1 transcription amount and AFT/Nor-1 (producing poison amount and nor-1 transcription amount ratio) respectively
Produce virulence comparative analysis
By result comparative analysis it was found that if single produce virulence size, mirror from nor-1 transcription amount to evaluate aspergillus flavus
It is unreliable to determine result.As the logarithm of aspergillus flavus strain Pc124-2 and Pc34-1, nor-1 gene expression amount copy number are respectively
7.85 ± 0.52 and 6.75 ± 0.58, two bacterial strain nor-1 gene expression doses are suitable；However the aspergillus flavus of Pc124-2 bacterial strain is malicious
Plain yield is 66.5 ± 4.93ng/mL, and the amount that Pc34-1 produces aflatoxin is only 19.69 ± 2.27ng/mL；In addition, CY1,
Aflatoxin generation is not detected in CY2, Pg28-1 and Pc321-1-3 bacterial strain, however, Pg28-1 and Pc321-1-3 expression
Nor-1 gene level even than production aflatoxin some bacterial strains it is higher.
But the ratio of aflatoxin yield and nor-1 gene expression dose is used to evaluate Aspergillus flavus and produces virulence
Result it is more reliable.In order to further verify the production virulence and nor-1 gene expression dose and AFT/Nor- of aspergillus flavus strain
Correlativity between 1.Poison amount is produced for ordinate, respectively with the logarithm and AFT/ of nor-1 gene expression amount with bacterial strain respectively
Nor-1 ratio is abscissa mapping.Aflatoxin yield and Nor-1 gene expression amount correlativity are as shown in Fig. 6-A, linearly
Regression equation are as follows: y=36.37x -196.21, the relative coefficient R that linear regression analysis generates2=0.693.However, aspergillus flavus
Output of toxin and AFT/Nor-1 ratio correlativity as shown in figure 6-b, equation of linear regression are as follows: y=10.14x -16.20, line
Property regression analysis generate relative coefficient R2=0.979.Aflatoxin yield generates extraordinary with AFT/Nor-1 ratio
Correlation.
Therefore, the present invention proposes that aspergillus flavus strain produces virulence and identifies equation are as follows: y=10.14x -16.20, wherein X value represents
AFT/Nor-1, Y are to produce virulence.
According to high yield toxic bacterial strain, poison amount > 150ng/mL is produced；Middle production poison: 50 < produce poison amount < 150ng/mL；Low yield poison: poison is produced
Amount < 50ng/mL；Do not produce poison: 0；Then it is obtained producing virulence identification range according to regression equation calculation:
High yield toxic bacterial strain: AFT/Nor-1 > 16.4；
Middle toxigenic bacterium strain: 6.5 < AFT/Nor-1 < 16.4；
Low yield toxic bacterial strain: 0 < AFT/Nor-1 < 6.5；
Not toxigenic bacterium strain: AFT/Nor-1=0.
AFT/Nor-1 (producing poison amount and nor-1 transcription amount ratio) identification produces the verifying of virulence result stability
Two, AFT/Nor-1 (producing poison amount and nor-1 transcription amount ratio) identification produces the verifying of virulence result stability
In order to further verify the stability that the identification of AFT/Nor-1 ratio produces virulence result, 5 plants of aspergillus flavus strains are distinguished
The experimental analysis between group in the group of aflatoxin yield and nor-1 genetic transcription amount is carried out.Experiment is on the same day in group
5 repetitions, production poison amount and nor-1 genetic transcription amount of 5 repetition average values as the batch bacterial strain is arranged in parallel.It is tested between group
Same date, each date are not a batch for setting 3, and 3 batches are arranged altogether.As a result such as the following table 5.
5 AFT/Nor-1 of table (producing poison amount and nor-1 transcription amount ratio) identification produces the verifying of virulence result stability
Note:1AFT: first batch aflatoxin yield；2Nor-1: second lot Nor-1 gene record amount；3Ratio:
Third batch AFT/Nor-1 ratio；*CV、#CV、&CV: being respectively detection aflatoxin yield, nor-1 base between different batches
Because of transcription amount and the AFT/Nor-1 ratio coefficient of variation；@: the coefficient of variation (CV) value is greater than 15%；
Aflatoxin yield, Nor-1 gene record amount are 5 repetition test average value in group in table.
We are it is not difficult to find that the yield and nor-1 gene of the aflatoxin of different batches culture detection turn from table
Record amount differs greatly.By taking bacterial strain 233-1 as an example, the yield of three batch detection aflatoxin is respectively 10.07,19.69 and
25.32ng/mL, different batches group difference coefficient are 42.00%.Furthermore bacterial strain IT-2, Pg56-1-2, N200 and 233-1 is produced
The amount of aflatoxin, different batches group difference coefficient are all larger than 17.00%.It can be seen that individually from aflatoxin
It is insecure that yield, which evaluates bacterial strain and produces virulence,.It similarly analyzes, nor-1 genetic transcription amount is found between different batches group, nor-1
Genetic transcription amount difference is also larger.It is unreliable that same confirmation only produces virulence from nor-1 genetic transcription amount characterization and evaluation bacterial strain.So
And the group difference coefficient of AFT/Nor-1 ratio is respectively less than 13%, has better stability.
To sum up illustrate, the method for being used to characterization and evaluation aflatoxicogenic strain and producing virulence proposed in the present invention, i.e.,
The identification of AFT/Nor-1 ratio is a method that more accurately and reliably characterization and evaluation aspergillus flavus strain produces virulence.
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation or change therefore amplified
It moves within still in the protection scope of the invention.
Sequence table
110 > Inst. of Oil Crops, Chinese Academy of Agriculture of <
120 > aflatoxin yield of < withNor-1The synchronous detection RT-PCR kit and its detection method of genetic transcription amount
160 > 6 of <
<210> 1
<211>18bp
<212> DNA
<213>artificial sequence
<400> 1
gtggtagcac aaactatg 18
<210> 2
<211>18bp
<212> DNA
<213>artificial sequence
<400> 2
ggctgcacag taataaac 18
<210> 3
<211>23bp
<212> DNA
<213>artificial sequence
<400> 3
ccgattcacc atctccagag aca 23
<210> 4
<211>20bp
<212> DNA
<213>artificial sequence
<400> 4
gtccaagcaa caggccaagt 20
<210> 5
<211>20bp
<212> DNA
<213>artificial sequence
<400> 5
tcgtgcatgt tggtgatggt 20
<210> 6
<211>18bp
<212> DNA
<213>artificial sequence
<400> 6
tgtcttgatc ggcgcccg 18
Claims (10)
1. a kind of aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount, which is characterized in that packet
It includes:
1) for expand surface display aflatoxin antiidiotype nano antibody bacteriophage institute released dna segment primer and
Probe: upstream primer Ph-F, nucleotide sequence is as shown in SEQ ID NO.1；Downstream primer Ph-R, nucleotide sequence such as SEQ ID
Shown in NO.2；Fluorescence probe Ph-probe, nucleotide sequence is as shown in SEQ ID NO.3；
2) for expanding the primer and probe of Tq-nor-1 gene: upstream primer Tq-nor1-F, nucleotide sequence such as SEQ ID
Shown in NO.4；Downstream primer Tq-nor1-R, nucleotide sequence is as shown in SEQ ID NO.5；Fluorescence probe Tq-probe, nucleosides
Acid sequence is as shown in SEQ ID NO.6；
3) universal probe qPCR premixed liquid, archaeal dna polymerase, MgCl2、dNTPs、H2O。
2. aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount according to claim 1,
Be characterized in that, the upstream and downstream primer: Ph-F, Ph-R, Tq-nor1-F, Tq-nor1-R are in PT-PCR amplification reaction system
Final concentration is 300~400nM.
3. aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount according to claim 1,
Be characterized in that, the fluorescence probe: final concentration of the Ph-probe and Tq-probe in PT-PCR amplification reaction system is 200
~400nM.
4. aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount according to claim 1,
It is characterized in that, the archaeal dna polymerase final dosage in PT-PCR amplification reaction system is 0.5U~1.0U, the MgCl2?
The end of final concentration of 1mM~2mM in PT-PCR amplification reaction system, the dNTPs in PT-PCR amplification reaction system is dense
Degree is 200uM~400uM.
5. being turned using any kit of Claims 1 to 44 with one step RT-PCR detection aflatoxin yield and Nor-1 gene
The method of record amount, which comprises the steps of:
(1) foundation of quantitative aflatoxin S type standard curve: the Immune competition stage of reaction, it is anti-in aflatoxin monoclonal
In the case that body package amount is certain, using the aflatoxin mark product and surface display aflatoxin antiidiotype of various concentration
The bacteriophage competitive binding aflatoxin monoclonal antibody of nano antibody, Immune competition after reaction, are incorporated into Huang Qu
The bacteriophage elution of surface display aflatoxin antiidiotype nano antibody on mould toxin monoclone antibody, various concentration
The corresponding bacteriophage for eluting different amounts of surface display aflatoxin antiidiotype nano antibody of aflatoxin mark product, elution
Bacteriophage in liquid understands released dna molecule during PCR is heated, and the DNA molecular of release is as the expansion in RT-PCR reaction
Increase target, each eluent is synchronized by RT-PCR amplified reaction using the kit respectively, is obtained after amplified reaction
Different Ct values, using Ct value as ordinate, carries out regression analysis, obtains using the logarithm of aflatoxin concentration as abscissa
The quantitative S type standard curve of aflatoxin；
(2) foundation of Nor-1 genetic transcription amount RT-PCR standard curve: by nor-1 gene DNA fragment Tq-nor1 known copy
Then several sample serial dilutions synchronizes each copy number Tq-nor1 using the kit respectively at different copy numbers
RT-PCR amplified reaction obtains different Ct values after amplified reaction, using the logarithm of Tq-nor1 copy number as abscissa,
Using Ct value as ordinate, regression analysis is carried out, the quantitation curves of nor-1 genetic transcription amount are obtained；
(3) aspergillus flavus strain is cultivated, takes Aspergillus flavus spore to carry out shaken cultivation, after culture, filters to take strain cultured solution
With Aspergillus flavus pompon；Aspergillus flavus poison after strain cultured solution is diluted certain multiple, in alternative steps (1) described immune response
Plain mark product participate in Immune competition reaction, the surface display being incorporated into aflatoxin monoclonal antibody after Immune competition reaction
The bacteriophage elution of aflatoxin antiidiotype nano antibody, the DNA molecular that eluent pnagus medius is discharged is as synchronization
The template of quantitative amplification aflatoxin in RT-PCR amplified reaction；Separately by after the drying of Aspergillus flavus pompon, it is simultaneously anti-to extract total serum IgE
Be transcribed into cDNA, by the cDNA dilute certain multiple after as quantitative amplification Nor-1 gene in same one step RT-PCR amplified reaction
Template；
(4) RT-PCR amplified reaction is synchronized using the DNA molecular of bacteriophage release and the cDNA as template, amplification is anti-
Two Ct values are obtained after answering, and two Ct values are substituted into the quantitative S type standard curve and nor-1 gene of aflatoxin respectively
The quantitation curves of transcription amount, conversion obtain aflatoxin concentration and nor-1 genetic transcription amount, determine aspergillus flavus poison with this
The ratio of plain yield and Nor-1 genetic transcription amount.
6. aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount according to claim 5,
It is characterized in that, the bacteriophage of the surface display aflatoxin antiidiotype nano antibody is bacteriophage VHH 2-5, the Huang
Aspertoxin monoclonal antibody is aflatoxin monoclonal antibody 1C11.
7. the method for detecting aflatoxin yield and Nor-1 genetic transcription amount with one step RT-PCR according to claim 5,
Be characterized in that, in the reaction system of the same one step RT-PCR amplified reaction, upstream and downstream primer: Ph-F, Ph-R, Tq-nor1-F,
The final concentration of Tq-nor1-R is 300~400nM；Fluorescence probe: the final concentration 200 of Ph-probe and Tq-probe~
400nM；The final dosage of archaeal dna polymerase: 0.5U~1.0U；MgCl2Final concentration: 1mM~2mM；DNTPs final concentration: 200uM~
400uM。
8. the method for detecting aflatoxin yield and Nor-1 genetic transcription amount with one step RT-PCR according to claim 5,
It is characterized in that, the reaction system of the same one step RT-PCR amplified reaction includes: universal 5 μ L of probe qPCR premixed liquid, Ph-F
0.1 μ L, 0.1 Ph-R μ L, 0.1 Ph-probe μ L, 2 μ L of bacteriophage template, 0.1 Tq-nor1-F μ L, 0.1 Tq-nor1-R μ
L, 0.1 μ L of Tq-probe, 1 μ L of Nor-1 gene template, 0.2 μ L of archaeal dna polymerase, MgCl20.8 μ L, 0.2 dNTPs μ L, add H2O
10 μ L of polishing.
9. the method for detecting aflatoxin yield and Nor-1 genetic transcription amount with one step RT-PCR according to claim 5,
It is characterized in that, the amplification condition of the same one step RT-PCR amplified reaction is 95 DEG C, 5min；95 DEG C, 10s, 60 DEG C of 30s, 40 are followed
Ring.
10. the method for detecting aflatoxin yield and Nor-1 genetic transcription amount with one step RT-PCR according to claim 5,
It is characterized in that, aflatoxin concentration range is in the quantitative S type standard curve of step (1) described aflatoxin
33.33ng/mL~1.69pg/mL, the lowest detection line LOD of aflatoxin are 0.018ng/mL；Step (2) described nor-1
Nor-1 gene copy number range is 10 in the quantitation curves of genetic transcription amount2~108。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811494322.0A CN109468367B (en) | 2018-12-07 | 2018-12-07 | RT-PCR (reverse transcription-polymerase chain reaction) kit for synchronously detecting yield of aflatoxin and Nor-1 gene transcription amount and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811494322.0A CN109468367B (en) | 2018-12-07 | 2018-12-07 | RT-PCR (reverse transcription-polymerase chain reaction) kit for synchronously detecting yield of aflatoxin and Nor-1 gene transcription amount and detection method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109468367A true CN109468367A (en) | 2019-03-15 |
| CN109468367B CN109468367B (en) | 2021-06-22 |
Family
ID=65675755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811494322.0A Active CN109468367B (en) | 2018-12-07 | 2018-12-07 | RT-PCR (reverse transcription-polymerase chain reaction) kit for synchronously detecting yield of aflatoxin and Nor-1 gene transcription amount and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109468367B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210189512A1 (en) * | 2018-12-07 | 2021-06-24 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Method for identifying and evaluating toxigenic capability of aflatoxigenic strain |
| CN113607949A (en) * | 2021-05-28 | 2021-11-05 | 中国农业科学院油料作物研究所 | Method for rapidly identifying and comparing relative abundance of toxin-producing fungi of aflatoxin in farmland |
| CN113721022A (en) * | 2021-09-07 | 2021-11-30 | 中国农业科学院油料作物研究所 | Method for rapidly identifying relative abundance of aflatoxin toxigenic bacteria in farmland and application thereof |
| CN113831396A (en) * | 2021-09-07 | 2021-12-24 | 中国农业科学院油料作物研究所 | Molecule for indicating aflatoxin toxigenicity of toxigenic bacteria and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993855A (en) * | 2010-08-05 | 2011-03-30 | 中国农业科学院油料作物研究所 | Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof |
| JP2014070038A (en) * | 2012-09-28 | 2014-04-21 | Univ Of Tokyo | AFLATOXIN PRODUCTION INHIBITOR AND PRODUCTION METHOD OF THE SAME, NORSOLORINIC ACID PRODUCTION INHIBITOR, mRNA EXPRESSION INHIBITOR, AND AFLATOXIN CONTAMINATION CONTROL METHOD |
| CN104152548A (en) * | 2014-07-07 | 2014-11-19 | 山东省农业科学院生物技术研究中心 | Biochip, detection kit and detection method for detecting mold for producing aflatoxin |
| CN108535376A (en) * | 2018-03-30 | 2018-09-14 | 中国农业科学院油料作物研究所 | The detection method of aflatoxin in a kind of vegetable protein beverage |
-
2018
- 2018-12-07 CN CN201811494322.0A patent/CN109468367B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993855A (en) * | 2010-08-05 | 2011-03-30 | 中国农业科学院油料作物研究所 | Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof |
| JP2014070038A (en) * | 2012-09-28 | 2014-04-21 | Univ Of Tokyo | AFLATOXIN PRODUCTION INHIBITOR AND PRODUCTION METHOD OF THE SAME, NORSOLORINIC ACID PRODUCTION INHIBITOR, mRNA EXPRESSION INHIBITOR, AND AFLATOXIN CONTAMINATION CONTROL METHOD |
| CN104152548A (en) * | 2014-07-07 | 2014-11-19 | 山东省农业科学院生物技术研究中心 | Biochip, detection kit and detection method for detecting mold for producing aflatoxin |
| CN108535376A (en) * | 2018-03-30 | 2018-09-14 | 中国农业科学院油料作物研究所 | The detection method of aflatoxin in a kind of vegetable protein beverage |
Non-Patent Citations (4)
| Title |
|---|
| A.ABDEL-HADI等: "Temporal monitoring of the nor-1(aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels.", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| MOHAMED N.HASSAN等: "Detection of aflatoxins by HPLC and the expression of biosynthetic nor-1 gene of aflatoxin and ocrA gene of ochratoxin.", 《BENHA VETERINARY MEDICAL JOURNAL》 * |
| XIANFENG REN等: "Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody-Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration", 《FRONTIERS IN MICROBIOLOGY》 * |
| 晏丽等: "荧光定量PCR检测发酵豆酱中黄曲霉毒素相关基因nor-1", 《食品与发酵工业》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210189512A1 (en) * | 2018-12-07 | 2021-06-24 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Method for identifying and evaluating toxigenic capability of aflatoxigenic strain |
| CN113607949A (en) * | 2021-05-28 | 2021-11-05 | 中国农业科学院油料作物研究所 | Method for rapidly identifying and comparing relative abundance of toxin-producing fungi of aflatoxin in farmland |
| CN113607949B (en) * | 2021-05-28 | 2023-06-27 | 中国农业科学院油料作物研究所 | Method for rapidly identifying and comparing relative abundance of toxigenic fungi of farmland aflatoxins |
| CN113721022A (en) * | 2021-09-07 | 2021-11-30 | 中国农业科学院油料作物研究所 | Method for rapidly identifying relative abundance of aflatoxin toxigenic bacteria in farmland and application thereof |
| CN113831396A (en) * | 2021-09-07 | 2021-12-24 | 中国农业科学院油料作物研究所 | Molecule for indicating aflatoxin toxigenicity of toxigenic bacteria and application thereof |
| CN113721022B (en) * | 2021-09-07 | 2023-06-27 | 中国农业科学院油料作物研究所 | Quick identification method for relative abundance of aflatoxin-producing bacteria in farmland and application thereof |
| CN113831396B (en) * | 2021-09-07 | 2023-12-19 | 中国农业科学院油料作物研究所 | Molecule for indicating toxicity of aflatoxin-producing bacteria and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109468367B (en) | 2021-06-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109557314A (en) | A method of virulence is produced for characterization and evaluation aflatoxicogenic strain | |
| CN109468367A (en) | Aflatoxin yield detection RT-PCR kit synchronous with Nor-1 genetic transcription amount and its detection method | |
| Nguyen et al. | The lung mycobiome: an emerging field of the human respiratory microbiome | |
| CN101570782B (en) | Detection kit and detection method for 8 species of pathogenic bacteria in dairy products | |
| CN105695631B (en) | Human immunodeficiency virus, hepatitis type B virus, Hepatitis C Virus Quick joint inspection kit and its preparation and application | |
| CN107619850A (en) | A kind of method of the quantitative detection vibrio alginolyticus based on aptamer | |
| CN103018453B (en) | A kind of chemical luminescence ELISA detection kit of QNS | |
| CN110373485A (en) | A kind of ureaplasma urealyticum, three joint inspection kit of chlamydia trachomatis and gonococcus | |
| CN110273026A (en) | Respiratory tract infection Multiple detection kit and detection method | |
| Ji et al. | Volatile metabolite profiling of wheat kernels contaminated by Fusarium graminearum | |
| CN102399753A (en) | Specific monoclonal antibody of Tilletia controversa Kuhn and immunofluorescence detection method | |
| Kantak et al. | Integration of DNA barcoding and nanotechnology in drug delivery | |
| Cook et al. | Quantitative PCR method to measure the fungal endophyte in locoweeds | |
| CN106596934B (en) | A kind of kit for detecting O-shaped foot and mouth disease virus | |
| CN101709087A (en) | Puccinia triticina f.sp.tritic monoclonal antibody and preparation method and application thereof | |
| CN107523632A (en) | Ustilaginoidea virens spore real-time quantitative ring mediated isothermal amplification detection method and kit | |
| CN101838703A (en) | Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot | |
| CN107132347A (en) | A kind of time-resolved fluorescence detection kit of 5 fluorouracil blood concentrations of real-time monitoring | |
| CN101985652B (en) | High-throughput molecular detection of Fusarium graminearum on carbendazim medicament-resistant gene frequency | |
| CN102181576B (en) | Primer, probe and method for detecting respiratory infectious disease pathogen by using liquid chip | |
| AU2021100496A4 (en) | A fast qualitative and quantitative kit for microorganisms and a fast qualitative and quantitative method | |
| CN107674058A (en) | Antiviral compound and preparation method and application | |
| Sher et al. | Dynamic nitrogen fixation in an aerobic endophyte of Populus | |
| Liu et al. | Quantitative detection of Ganodermati lucidum immunomodulatory protein-8 by a peptide-antigen-antibody sandwich ELISA | |
| CN107488594A (en) | One plant of new Penicillium notatum and its metabolite pacify him and intend sour A |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |