CN113293225B - Primer probe combination capable of specifically identifying aspergillus, penicillium and fusarium and application thereof - Google Patents
Primer probe combination capable of specifically identifying aspergillus, penicillium and fusarium and application thereof Download PDFInfo
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Classifications
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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
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a primer probe combination capable of specifically identifying aspergillus, penicillium and fusarium and application thereof. According to the invention, aspergillus, penicillium and fusarium are used as targets, and a universal primer is designed, and a specific probe is designed to perform qPCR experiment on the basis of determining the primer universality, so that amplification curves and standard curves of aspergillus, penicillium and fusarium are obtained, and qualitative and quantitative detection of aspergillus, penicillium and fusarium contained in nuts is realized.
Description
Technical Field
The invention relates to the technical field of food safety biology, in particular to a primer probe combination capable of specifically identifying aspergillus, penicillium and fusarium and application thereof.
Background
With the development of economy and the increase of consumption level, nut foods are becoming daily consumer products, and the quality demands of consumers on nut foods are also increasing. However, in recent years, the phenomenon that mould in the market supervision spot check nut food exceeds standard is frequent, so that economic loss of the food industry is caused, and potential health risks are brought to consumers. Mould is commonly called as filamentous fungi, toxic metabolic mycotoxin is easy to generate in foods polluted by mould, acute or chronic poisoning of human bodies can be caused, liver, kidney, nerve tissue, hematopoietic tissue, skin tissue and the like of organisms are damaged, and meanwhile, the fungi are easy to disperse in air due to the spore generating capability of the fungi, and the fungi appear at any time or stage in a food processing chain. Among the common moulds in nuts are Aspergillus, penicillium and Fusarium, and how to detect moulds rapidly in the early stage of production is a hot spot problem which needs to be solved in the industry.
The current common methods for detecting the mould are a traditional culture method and a biochemical detection technology. The traditional culture method, namely national standard method-GB 4789.15-2016, is used for detecting mould and yeast counts in food safety national standard food microbiology, has accurate results, longer detection period and complicated operation steps, and is not suitable for the requirements of rapid and efficient detection. Biochemical detection techniques are techniques in which the detection of species and quantities is performed by specific reactions between metabolites produced by the mould during growth and reproduction and specific substrates. The common detection methods are a rapid test strip method and an ATP bioluminescence technique detection method. The method is simple to operate and low in cost, but the accuracy of the detection result is poor.
The PCR detection has the advantages of short detection time, high sensitivity, strong specificity and the like, but the improvement of the accuracy and the specificity of the detection result is one of the difficulties and the key points of biological detection. Furthermore, PCR detection cannot reach quantification but can only stay in qualitative phase. And meanwhile, the aerosol pollution caused by uncovering in the detection process can cause false positive of a detection result. In the PCR product identification link, the coloring agent used in agarose gel electrophoresis is extremely toxic, and the concentration and time of glue preparation can influence the final detection result, so that the technology of operators is highly required.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primer probe combination capable of specifically identifying aspergillus, penicillium and fusarium and application thereof. The invention takes aspergillus, penicillium and fusarium as targets, designs universal primers through DNAMAN software comparison sequences, oligo 7 and other software, designs specific probes on the basis of determining the universality of the primers to carry out qPCR test, obtains amplification curves and standard curves of the aspergillus, penicillium and fusarium, and realizes qualitative and quantitative detection of the aspergillus, penicillium and fusarium contained in nuts.
The technical scheme of the invention is as follows:
a primer probe combination capable of specifically identifying aspergillus, penicillium and fusarium, wherein the primer is a universal primer capable of specifically identifying aspergillus, penicillium and fusarium, and comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the probes include Aspergillus molecular beacon probes, penicillium molecular beacon probes, and Fusarium molecular beacon probes.
Further, the nucleotide sequence of the aspergillus molecular beacon probe is shown as SEQ ID No. 3.
Further, the nucleotide sequence of the penicillium molecular beacon probe is shown as SEQ ID No. 4.
Further, the nucleotide sequence of the fusarium molecular beacon probe is shown as SEQ ID No. 5.
Further, the 5 'end of the nucleotide sequence of the probe is marked with a fluorescent recognition group, and the 3' end is marked with a fluorescent quenching group.
Further, the fluorescent recognition group is one or more of FAM, VIC, NED; the fluorescence quenching group is one or more of Dabcyl, BHQ1 and BHQ 2.
A kit comprises the primer probe combination.
Further, the detection limit of the kit on aspergillus is 2.5×10 -2 ng/mL, linear detection range of 2.5×10 -2 ～1.2*10 2 ng/mL; the detection limit of the penicillium is 8.7x10 -3 ng/mL, linear detection range of 8.7X10 -3 93ng/mL; the detection limit of fusarium is 5.6x10 -3 ng/mL, linear detection range of 5.6x10 -3 ～110ng/mL。
Use of the kit for simultaneous detection of aspergillus, penicillium and fusarium in nuts.
Further, a method for simultaneously detecting aspergillus, penicillium, and fusarium in nuts using the kit comprises the steps of:
(1) Establishment of amplification curves of different bacteria:
extracting genome of standard sample of Aspergillus, penicillium and Fusarium respectively, adding primer probe combination, preparing 20 μl qPCR system, setting qPCR program to perform amplification experiment, and detecting fluorescence response of bacteria in sample in annealing stage to obtain amplification curve of each bacteria;
(2) Establishing standard curves of aspergillus, penicillium and fusarium:
respectively preparing standard substance genomes of aspergillus, penicillium and fusarium with different concentrations, adding a primer probe combination, preparing a 20 mu l qPCR system, setting a qPCR program to perform an amplification experiment, and respectively establishing standard curves corresponding to the aspergillus, penicillium and fusarium by detecting fluorescence responses of standard substance solutions with different concentrations in an annealing stage;
(3) Detection of aspergillus, penicillium and fusarium contained in nuts:
pretreating a nut sample, culturing the nut sample in a liquid culture medium, taking hypha liquid nitrogen in the culture medium for breaking, extracting a sample genome from the broken tissue by a kit method, adding a primer probe combination, preparing a 20 mu lqPCR system, setting a qPCR program for an amplification experiment, judging the genus of the nut sample by detecting the fluorescence response of the nut sample in an annealing stage, and calculating the genome concentration of aspergillus, penicillium and fusarium contained in the nut by combining different Ct values and corresponding standard curves.
The beneficial technical effects of the invention are as follows:
(1) According to the invention, through comparing different bacteria gene sequences in NCBI gene sequence library, universal primers of Aspergillus, penicillium and Fusarium are designed at a conservation site, so that the universal detection of common mould in nuts is ensured.
(2) The invention ensures the specificity of detecting aspergillus, penicillium and fusarium in nuts by designing the molecular beacon probes of different bacteria of aspergillus, penicillium and fusarium which are common in nuts.
(3) The invention carries out DNA extraction and qPCR amplification on different bacteria in nuts, realizes multiple qualitative and quantitative analysis, has simple and quick operation, and can complete the whole detection process within a few hours.
(4) The whole detection process does not need to cover the cover, so that aerosol pollution caused by cover opening in common PCR detection is avoided; fluorescent detection is used for replacing the coloring agent, and the detection process is safer.
Drawings
FIG. 1 is a diagram of a specific PCR amplification of a mould to be detected according to the invention.
In the figure: lane 1 is 10k bp ladder marker; lane 2 aspergillus niger (Aspergillus niger CICC 2487); lane 3 aspergillus flavus (Aspergillus flavus CICC 2219); lane 4 is aspergillus peak (Aspergillus nomius ACCC 32558); lane 5 is penicillium green (Penicillium viridicatum CICC 4029); lane 6 is fusarium oxysporum (Fusarium oxysporum CICC 41029).
FIG. 2 is a graph showing the amplification of Aspergillus fungi according to example 2 of the present invention.
FIG. 3 is a graph showing the amplification of Penicillium fungus according to example 2 of the present invention.
FIG. 4 is a graph showing the amplification of Fusarium fungi according to example 2 of the present invention.
FIG. 5 shows a single qPCR sensitivity test and standard curve for Aspergillus fungi according to example 2 of the present invention.
FIG. 6 shows the sensitivity detection and standard curve of the Penicillium fungus single qPCR according to example 2 of the present invention.
FIG. 7 is a single qPCR sensitivity test and standard curve for Fusarium fungi of example 2 of the present invention.
FIG. 8 shows a multiplex qPCR sensitivity assay and standard curve for Aspergillus fungi according to example 3 of the present invention.
FIG. 9 shows a multiplex qPCR sensitivity assay and standard curve for Penicillium fungus according to example 3 of the present invention.
FIG. 10 is a multiplex qPCR sensitivity assay and standard curve for Fusarium fungi of example 3 of the present invention.
FIG. 11 is a diagram showing the mixed genus multiplex qPCR specific assay of example 3 of the present invention.
In the figure: a: aspergillus niger, penicillium viridis and Fusarium oxysporum mixed DNA; b: aspergillus peak, penicillium viridis and Fusarium oxysporum mixed DNA; c: aspergillus flavus, penicillium viridis and Fusarium oxysporum mixed DNA; d: aspergillus niger, aspergillus flavus and Aspergillus flavus mix DNA.
FIG. 12 is a graph showing the results of the detection of Aspergillus, penicillium and Fusarium species in nuts according to example 4 of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
The invention includes, but is not limited to, the following examples, any equivalent substitutions or refusal modifications made under the spirit and principles of the invention are to be considered as being within the scope of the present invention.
In the following examples, primers and probes were synthesized by Shanghai Biotechnology services Co., ltd:
the nucleotide sequence of the upstream primer is (shown as SEQ ID No. 1):
5’-GGCATCGATAGAGAACGCAGC-3’
the nucleotide sequence of the downstream primer is (as shown in SEQ ID No. 2):
5’-CCTACCTGACTCGAGGTCAAC-3’
the nucleotide sequence of the aspergillus molecular beacon probe is (shown as SEQ ID No. 3):
5’-CGTCCGATCCTCAGGCGTATGGACG-3’
the nucleotide sequence of the penicillium molecular beacon probe is (shown as SEQ ID No. 4):
5’-CGTCAGTGTGTTGGCGCCCGTCTGACG-3’
the nucleotide sequence of the fusarium molecular beacon probe is (shown as SEQ ID No. 5):
5’-CGTCGCCGTTAAACCCACACTTCTGACGACG-3’
the nucleotide sequences of the aspergillus, penicillium and fusarium molecular beacon probes are respectively combined with fluorescent recognition groups at the 5 'ends and fluorescent quenching groups at the 3' ends, wherein the fluorescent recognition groups are one or more of FAM, VIC, NED, and the fluorescent quenching groups are one or more of Dabcyl, BHQ1 and BHQ 2.
The aspergillus, penicillium and fusarium used in the invention are specifically aspergillus niger (Aspergillus niger CICC 2487), aspergillus flavus (Aspergillus flavus CICC 2219), penicillium viridis (Penicillium viridicatum CICC 4029) and fusarium oxysporum (Fusarium oxysporum CICC 41029) which are all purchased from China industry microbiological culture collection center, and aspergillus peak collection (Aspergillus nomius ACCC 32558) is purchased from China industry microbiological culture collection center.
Example 1 design and verification of primers and probes
(1) The genome DNA of the tested strain is extracted by adopting a kit method, and the specific method is as follows:
taking hypha of aspergillus flavus in a liquid culture medium, and grinding and crushing fungus tissues in liquid nitrogen; taking not more than 50mg of ground fungal tissue and placing into a 1.5ml microcentrifuge tube; 400 μl LE Buffer and 4 μl RNase A at 20mg/ml were added and mixed well; incubate at 65deg.C for 30min, vortex oscillating centrifuge tube every 10 min; adding 130 μl of DA Buffer, mixing, and standing in ice bath for 5min; centrifuging at 14000xg for 3min; transferring the supernatant to a new 1.5ml centrifuge tube; adding 750 μl of E Binding Buffer, and mixing uniformly; transferring the mixed liquid to Spin column, centrifuging at 6000xg for 1min, and discarding the liquid in the liquid receiving pipe; adding 500 μl of G Binding Buffer into Spin column, centrifuging at 10000xg for 30s, and discarding liquid in the liquid receiving tube; adding 600 μl of Wash Buffer into Spin column, centrifuging at 10000xg for 30s, discarding liquid in the liquid receiving tube, and repeating the steps for one time; centrifuging the Spin column at 10000xg for 1min again, and transferring the Spin column to a new 1.5ml centrifuge tube; 100. Mu.l to 200. Mu.l of the Elutation Buffer was added to Spin column and incubated at room temperature for 1min; centrifuging at 12000Xg for 1min, discarding the DNA of Aspergillus flavus contained in a Spin column 1.5ml centrifuge tube, preserving at-20deg.C, and similarly obtaining DNA of Aspergillus niger, aspergillus peak, penicillium viridis and Fusarium oxysporum, and preserving at-20deg.C.
(2) Design of primers and probes
The ITS (Intemal Transcribed Spacer) sequences of Aspergillus, penicillium and Fusarium are aligned by DNAMAN software, and a common conserved sequence of Aspergillus, penicillium and Fusarium is searched for and universal primer design is performed. And comparing the specificity of the corresponding sequences in an NCBI database, comparing the secondary structure through NUPACK, adjusting the base sequence of the stem so that the probe presents a stem loop structure, and finally comparing the annealing temperature so that the probe can be combined onto the target sequence before the primer in the annealing stage to obtain the aspergillus, penicillium and fusarium specific probes.
(3) Verification of primers and probes
And (3) primer verification:
50. Mu.l of a PCR reaction system comprising 5. Mu.l of 10 XPCR buffer (Mg-containing 2+ ) 4. Mu.l dNTP, 0.4. Mu.l Taq DNA polymerase (Shanghai Biotechnology engineering services Co., ltd.), 1. Mu.l each of the upstream primer and the downstream primer, 5. Mu.l of the mixed DNA template of Aspergillus, penicillium and Fusarium extracted in the step (1), 33.6. Mu.l of sterile ultrapure water. The amplification procedure was: after denaturation at 94 ℃ for 5min, the mixture enters a cycle of 30 cycles of 94 ℃ for 1min, 57 ℃ for 45s and 72 ℃ for 45 s. 2.5. Mu.l of the PCR product was electrophoresed on a 1% agarose gel, stained with GelRed, and the result was determined under an ultraviolet lamp according to the band size. If the product with the length of 300bp can be specifically amplified, the designed primer can be judged to be used for detecting the corresponding genus, and the PCR amplification result is shown in figure 1.
And (3) probe verification:
20. Mu.l of qPCR reaction system comprising qPCR probe Mix 10. Mu.l (purchased from Shanghai Biyun Biotechnology Co., ltd.), 10. Mu.l of each of the primers and 0.4. Mu.l of each of the 10. Mu.M probes (synthesized by Shanghai Biotechnology engineering Co., ltd.) was used for verifying Aspergillus, aspergillus probes were used for verifying Penicillium, fusarium probes were used for verifying Fusarium, and 2. Mu.l of mixed DNA of Aspergillus, penicillium and Fusarium extracted in step (1) was used for 6.4. Mu.l of sterile ultrapure water. The amplification procedure was: denaturation at 95℃for 3min; fluorescence was detected during the annealing phase for a total of 40 cycles of 15s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃.
The nucleotide sequences of the primers and probes designed in the application are as follows:
the nucleotide sequence of the upstream primer is (shown as SEQ ID No. 1):
5’-GGCATCGATAGAGAACGCAGC-3’
the nucleotide sequence of the downstream primer is (as shown in SEQ ID No. 2):
5’-CCTACCTGACTCGAGGTCAAC-3’
the nucleotide sequence of the aspergillus molecular beacon probe is (shown as SEQ ID No. 3):
5’-CGTCCGATCCTCAGGCGTATGGACG-3’
the nucleotide sequence of the penicillium molecular beacon probe is (shown as SEQ ID No. 4):
5’-CGTCAGTGTGTTGGCGCCCGTCTGACG-3’
the nucleotide sequence of the fusarium molecular beacon probe is (shown as SEQ ID No. 5):
5’-CGTCGCCGTTAAACCCACACTTCTGACGACG-3’
the nucleotide sequences of the aspergillus, penicillium and fusarium molecular beacon probes are respectively combined with fluorescent recognition groups at the 5 'ends and fluorescent quenching groups at the 3' ends, wherein the fluorescent recognition groups are one or more of FAM, VIC, NED, and the fluorescent quenching groups are one or more of Dabcyl, BHQ1 and BHQ 2.
Example 2 Single real-time qPCR detection
(1) Extraction of genome
Aspergillus niger, aspergillus flavus, aspergillus foetidus, penicillium viridis and Fusarium oxysporum genomes were extracted, respectively, according to the method described in example 1, step (1). The concentration of the obtained Aspergillus niger genome was 7.2 ng/. Mu.l; aspergillus flavus concentration was 7.2 ng/. Mu.l; aspergillus peak genome concentration was 6.9 ng/. Mu.l; the concentration of the genome of the penicillium green is 3.3 ng/. Mu.l; fusarium oxysporum genome concentration was 14 ng/. Mu.l.
(2) Establishment of single qPCR system and amplification curve
Building an aspergillus qPCR system and an amplification curve:
20. Mu.l of qPCR reaction system comprising qPCR probe Mix 10. Mu.l (purchased from Shanghai Biyun Biotechnology Co., ltd.), 10. Mu.M primer each 0.6. Mu.l and 10. Mu.M Aspergillus probe 0.4. Mu.l (synthesized by Shanghai Biotechnology services Co., ltd.) of primer probe kit, the above 5 kinds of mold mixed genomic DNA 2. Mu.l, 6.4. Mu.l of sterile ultrapure water.
The amplification procedure was: denaturation at 95℃for 3min; the fluorescence was detected at 95℃for 15s,60℃for 30s, and 72℃for 30s for a total of 40 cycles during the annealing phase to obtain an amplification curve. 3 replicates were run per experiment.
As shown in FIG. 2, when only Aspergillus probes were used, amplification curves were present for Aspergillus niger, aspergillus flavus and Aspergillus aculeatus, and amplification curves were not obtained for Penicillium viridis and Fusarium oxysporum, so that the single qPCR system had excellent specificity for Aspergillus.
Establishment of penicillium qPCR system and amplification curve:
the Aspergillus probe in the qPCR system is changed into the Penicillium probe, and other parameters are the same as those of the Aspergillus qPCR system, as shown in FIG. 3, only the amplification curve of the Penicillium viridis can be obtained, so that the single qPCR system has excellent specificity to the Penicillium.
Creation of fusarium qPCR system and amplification curve:
the Aspergillus probe in the qPCR system is changed into the Fusarium probe, and other parameters are the same as those of the Aspergillus qPCR system, as shown in FIG. 4, only the amplification curve of Fusarium oxysporum can be obtained at the moment, so that the single qPCR system has excellent specificity to Fusarium.
(3) Establishment of a Standard Curve
Establishing an aspergillus standard curve:
20 μl of qPCR reaction system comprises qPCR probe Mix 10 μl (purchased from Shanghai Biyun biotechnology Co., ltd.), 10 μl of each of 10 μM primers and 0.4 μl (synthesized by Shanghai Biotechnology services Co., ltd.) of each of the Aspergillus probes, and 2 μl of mixed genomic DNA of Aspergillus niger, penicillium viridis and Fusarium oxysporum extracted in step (1) at different concentrations, and 6.4 μl of sterile ultrapure water. The different concentrations are 8 diluted concentrations obtained by 10-time gradient dilution of the mixed genome DNA of the aspergillus niger, the penicillium harbouring and the fusarium oxysporum extracted in the step (1), and 3 concentrations are parallel.
The amplification procedure was: denaturation at 95℃for 3min; the temperature of 95 ℃ is 15s, the temperature of 60 ℃ is annealed for 30s, the temperature of 72 ℃ is 30s, total time is 40 cycles, fluorescence is detected in the annealing stage, and a standard curve is established according to the concentration and the fluorescence intensity.
The sensitivity and standard curve of the Aspergillus are shown in FIG. 5, the standard curve is y= -3.25x+20.92, R 2 =0.9974, amplification efficiency e=10 -1/k -1＝10 -1/-3.2501 -1＝2.031，EPercent=e=100% =103.1%. At a genome concentration of A.niger of 10 -4 The ng/. Mu.l can still detect positive, has strong detection sensitivity and can be used for quantitative analysis.
Establishment of a penicillium standard curve:
the Aspergillus probe in the qPCR system is changed into the Penicillium probe, other parameters are the same as those of the Aspergillus qPCR system, and the Penicillium standard curve is established according to the method for establishing the Aspergillus standard curve.
FIG. 6 shows the sensitivity and standard curve of the Penicillium single qPCR, and as can be seen from FIG. 6, the standard curve of Penicillium is y= -3.45x+25.48, R 2 0.9991, amplification efficiency E% = 95.0% at a penicillium emerald genome concentration of 10 -3 The ng/. Mu.l can still detect positive, has strong detection sensitivity and can be used for quantitative analysis. Creation of Fusarium standard curve:
the Aspergillus probe in the qPCR system is changed into the Fusarium probe, other parameters are the same as those of the Aspergillus qPCR system, and the Fusarium standard curve is established according to the method for establishing the Aspergillus standard curve.
FIG. 7 shows the sensitivity and standard curve of the single qPCR for Fusarium fungi, wherein the standard curve of Fusarium is y= -3.36x+20.08, R is shown in FIG. 7 2 0.9932, amplification efficiency E% = 98.4% at a fusarium oxysporum genome concentration of 10 - 4 The ng/. Mu.l can still detect positive, has strong detection sensitivity and can be used for quantitative analysis.
Example 3 multiplex real-time PCR detection
(1) Extraction of genome
Aspergillus niger, aspergillus flavus, aspergillus foetidus, penicillium viridis and Fusarium oxysporum genomes were extracted, respectively, according to the method described in example 1, step (1). The concentration of the obtained Aspergillus niger genome was 7.2 ng/. Mu.l; aspergillus flavus concentration was 7.2 ng/. Mu.l; aspergillus peak genome concentration was 6.9 ng/. Mu.l; the concentration of the genome of the penicillium green is 3.3 ng/. Mu.l; fusarium oxysporum genome concentration was 14 ng/. Mu.l.
(2) Establishment of multiplex real-time qPCR system and amplification curve
20. Mu.l of qPCR reaction system including a primer probe kit composed of qPCR probe Mix 10. Mu.l (purchased from Shanghai Biyun Biotechnology Co., ltd.), 10. Mu.l of each of the primers 0.6. Mu.l and 10. Mu.M of Aspergillus probe 0.4. Mu.l, 10. Mu.M of Penicillium probe 0.4. Mu.l, 10. Mu.M of Fusarium probe 0.4. Mu.l (synthesized by Shanghai Biotechnology Co., ltd.), DNA 2. Mu.l, 6.4. Mu.l of sterile ultrapure water. Wherein the DNA is mixed DNA of Aspergillus niger, penicillium viridis and Fusarium oxysporum; aspergillus peak, penicillium viridis and Fusarium oxysporum mixed DNA; aspergillus flavus, penicillium viridis and Fusarium oxysporum mixed DNA; aspergillus niger, aspergillus flavus and Aspergillus flavus mix DNA.
The amplification procedure was: denaturation at 95℃for 3min; fluorescence was detected during the annealing phase for a total of 40 cycles of 15s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃.
The established multiplex qPCR system can detect mixed DNA of Aspergillus niger, penicillium viridis and Fusarium oxysporum; aspergillus peak, penicillium viridis and Fusarium oxysporum mixed DNA; aspergillus flavus, penicillium viridis and Fusarium oxysporum mixed DNA; aspergillus niger, aspergillus flavus and Aspergillus flavus mix DNA. As shown in FIG. 11, when a plurality of target bacterial genomes are present in the system, the target fragments can be amplified, and corresponding fluorescent signals can be generated.
(3) Establishment of a Standard Curve
20. Mu.l of qPCR reaction system comprising qPCR probe Mix 10. Mu.l (purchased from Shanghai Biyun Biotechnology Co., ltd.), 10. Mu.M primer each 0.6. Mu.l and 10. Mu.M Aspergillus probe 0.4. Mu.l, 10. Mu.M Fusarium probe 0.4. Mu.l (synthesized by Shanghai Biotechnology Co., ltd.) primer probe kit, 2. Mu.l of mixed genomic DNA of Aspergillus niger, penicillium viridis and Fusarium oxysporum extracted in step (1) of different concentrations, 6.4. Mu.l of sterile ultrapure water. The different concentrations are that the concentration of the mixed genome DNA of the aspergillus niger, the penicillium harbouring and the fusarium oxysporum extracted in the step (1) is diluted into 8 concentrations according to a 10-time gradient, and 3 concentrations are parallel.
The amplification procedure was: denaturation at 95℃for 3min; fluorescence was detected during the annealing phase for a total of 40 cycles of 15s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃. By detecting the fluorescence responses of the bacteria with different DNA concentrations in the annealing stage, standard curves corresponding to Aspergillus, penicillium and Fusarium are respectively established.
FIG. 8 shows the multiplex qPCR sensitivity test and standard curve for Aspergillus fungi, where the Aspergillus standard curve is y= -3.62x+21.38, R 2 0.9937, amplification efficiency E% = 88.9%, x is lg (ng/PCR system), y is Ct value, and the linear range of detection is 2.5X10 -2 -1.2×10 2 ng/mL。
FIG. 9 shows a multiplex qPCR sensitivity assay and standard curve for Penicillium fungi. As shown in fig. 9, the standard curve for penicillium detection is y= -3.49x+21.46, r 2 0.9922 amplification efficiency E% = 93.5%, x is lg (ng/PCR system), y is Ct value, and the linear range of detection is 8.7x10 -3 -93ng/mL。
FIG. 10 is a multiplex qPCR sensitivity assay and standard curve for Fusarium fungi of example 3 of the present invention. As shown in fig. 10, the standard curve for fusarium detection is y= -3.39x+19.07, r 2 Amplification efficiency E% = 97.1%, x is l g (ng/PCR system), y is Ct value, and the linear range of detection is 5.6x10 -3 -110ng/mL。
Example 4 sample detection
(1) Sample processing
25g of sample nuts are taken, added to 225mL of sterilized water, shaken for 10min, 200. Mu.l of the shaken solution is taken and added to 100mL of potato liquid medium, and the mixture is cultured for 40h at 28 ℃.
(2) Sample DNA extraction
Taking hyphae in the culture medium of the step (1), extracting the sample genome DNA according to the method of the step (1) of the example 1.
(3) Nut sample qPCR system establishment and detection
20. Mu.l of qPCR reaction system including qPCR probe Mix 10. Mu.l (purchased from Shanghai Biyun Biotechnology Co., ltd.), 10. Mu.M primer each 0.6. Mu.l and 10. Mu.M Aspergillus probe 0.4. Mu.l, 10. Mu.M Fusarium probe 0.4. Mu.l (synthesized by Shanghai Biotechnology Co., ltd.), and 2. Mu.l of the genomic DNA extracted in step (2) and 6.4. Mu.l of sterile ultrapure water.
The amplification procedure was: denaturation at 95℃for 3min; fluorescence was detected during the annealing phase for a total of 40 cycles of 15s at 95 ℃, 30s at 60 ℃ and 30s at 72 ℃. And calculating genome concentrations of aspergillus, penicillium and fusarium contained in the nuts by combining different Ct values with corresponding standard curves. As shown in FIG. 12, the presence of amplified bands of Aspergillus and Penicillium in the nuts revealed that Aspergillus and Penicillium were the main contaminating bacteria, the Aspergillus content in the nuts was 8.29 ng/. Mu.l, the Penicillium content was 4.67 ng/. Mu.l, and Fusarium was not detected.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> a primer probe combination capable of specifically recognizing Aspergillus, penicillium and Fusarium and application thereof
<130> 1
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> upstream primer sequence
<400> 1
ggcatcgata gagaacgcag c 21
<210> 2
<211> 21
<212> DNA
<213> downstream primer sequence
<400> 2
cctacctgac tcgaggtcaa c 21
<210> 3
<211> 25
<212> DNA
<213> Aspergillus molecular beacon probe sequences
<400> 3
cgtccgatcc tcaggcgtat ggacg 25
<210> 4
<211> 27
<212> DNA
<213> Penicillium molecular beacon Probe sequence
<400> 4
cgtcagtgtg ttggcgcccg tctgacg 27
<210> 5
<211> 31
<212> DNA
<213> Fusarium molecular beacon probe sequences
<400> 5
cgtcgccgtt aaacccacac ttctgacgac g 31
Claims (5)
1. Use of a kit comprising a combination of primer probes capable of specifically recognizing aspergillus, penicillium, fusarium, characterized in that the kit is used for simultaneous detection of aspergillus, penicillium and fusarium in nuts;
a method for simultaneously detecting aspergillus, penicillium, and fusarium in nuts using the kit comprising the steps of:
(1) Establishing amplification curves of different bacteria;
(2) Establishing a standard curve of aspergillus, penicillium and fusarium;
(3) Detecting Aspergillus, penicillium and Fusarium contained in the nuts;
the primer is a universal primer capable of specifically identifying aspergillus, penicillium and fusarium, and comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2; the probe comprises an aspergillus molecular beacon probe, a penicillium molecular beacon probe and a fusarium molecular beacon probe;
the nucleotide sequence of the aspergillus molecular beacon probe is shown as SEQ ID No. 3;
the nucleotide sequence of the penicillium molecular beacon probe is shown as SEQ ID No. 4;
the nucleotide sequence of the fusarium molecular beacon probe is shown as SEQ ID No. 5.
2. The use according to claim 1, wherein the probe has a fluorescent recognition group at the 5 'end and a fluorescent quenching group at the 3' end of the nucleotide sequence.
3. The use according to claim 2, wherein the fluorescent recognition groups are one or more of FAM, VIC, NED; the fluorescence quenching group is one or more of Dabcyl, BHQ1 and BHQ 2.
4. A kit comprising the primer probe combination of claim 1.
5. The kit according to claim 4, wherein the detection limit of the kit to aspergillus is 2.5 x 10 -2 ng/mL, linear detection range of 2.5×10 -2 ~1.2*10 2 ng/mL; the detection limit of the penicillium is 8.7x10 -3 ng/mL, linear detection range of 8.7X10 -3 93ng/mL; the detection limit of fusarium is 5.6x10 -3 ng/mL, linear detection range of 5.6x10 -3 ~110 ng/mL。
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| CN111100910A (en) * | 2018-10-26 | 2020-05-05 | 中国检验检疫科学研究院 | Primer probe and kit for qualitatively detecting aspergillus with toxin-producing potential |
| CN111100941A (en) * | 2018-10-25 | 2020-05-05 | 中国检验检疫科学研究院 | Primer probe and method for rapidly detecting penicillium with toxigenic potential |
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| CN111100941A (en) * | 2018-10-25 | 2020-05-05 | 中国检验检疫科学研究院 | Primer probe and method for rapidly detecting penicillium with toxigenic potential |
| CN111100910A (en) * | 2018-10-26 | 2020-05-05 | 中国检验检疫科学研究院 | Primer probe and kit for qualitatively detecting aspergillus with toxin-producing potential |
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