CN113684303A - Primer combination and kit for detecting mould in nuts and detection method for mycotoxin-producing fungus species in nuts - Google Patents
Primer combination and kit for detecting mould in nuts and detection method for mycotoxin-producing fungus species in nuts Download PDFInfo
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Abstract
The invention discloses a primer combination and a kit for detecting mould in nuts and a method for detecting the type of mycotoxin-producing fungi in nuts.
Description
Technical Field
The invention belongs to the technical field of food safety biology, and particularly relates to a primer combination and a kit for detecting mould in nuts and a method for detecting the type of mycotoxin-producing fungi in nuts.
Background
Mycotoxins are low molecular weight compounds produced by filamentous fungi, wherein trichothecenes, fumonisins, aflatoxins, ochratoxins and the like are common, and can cause different degrees of harm to human health and huge economic loss to the food industry. These toxic compounds are produced primarily by aspergillus, penicillium and fusarium species. Aspergillus flavus is reported in mildew pollution of cashew nuts, pistachios, chestnuts, almonds and the like, and is a main pollutant for causing mildew of nuts. In addition, the study reports that the penicillium is one of the main pathogenic bacteria causing the mildewing of the walnuts during the storage process. The detection of mycotoxins and mycotoxin-producing fungi is critical to food safety. The identification of fungi is expensive and time consuming. Therefore, the development of a rapid and simple method for detecting and identifying the presence of toxin-producing fungi would be beneficial for product pretreatment and for making important decisions about specific mycotoxin analysis.
The conventional methods for detecting the mould are a traditional culture method and a biochemical detection technology. The traditional culture method, namely the national standard method GB 4789.15-2016 (national food safety Standard food microbiology inspection) for mold and yeast counting, has accurate result, long detection period and complicated operation steps, and is not suitable for the requirement of rapid and efficient detection. The biochemical detection technology is to detect the species and quantity of the mold through the specific reaction between the metabolites and specific substrates generated in the growth and reproduction of the mold. The commonly used detection methods include a rapid test strip method and an ATP bioluminescence technology detection method. The rapid test strip method is simple to operate and low in cost, but the accuracy of the detection result is not good; the ATP bioluminescence method only detects living microorganisms, is difficult to distinguish the types of the microorganisms and is easy to cause false positive of detection.
Disclosure of Invention
In order to solve the technical problems, the invention provides a primer combination and a kit for detecting mould in nuts and a method for detecting the type of mycotoxin-producing fungi in nuts.
The technical scheme adopted by the invention is as follows:
a primer combination for nut mildew detection, the primer combination comprising:
amplifying a primer pair of a beta-tubulin region of an Aspergillus niger genome;
amplifying a primer pair of an alpha-amylase region of an aspergillus flavus genome;
amplifying a primer pair of an alpha-amylase region of an aspergillus peaking genome;
amplifying a primer pair of a beta-tubulin region of the penicillium genome;
primer pairs for amplifying the RPB2 region of fusarium genome.
The GenBank accession numbers of the Aspergillus niger genome, the Aspergillus flavus genome, the Aspergillus oryzae genome, the Penicillium genome and the Fusarium genome are EU982068.1, DQ467916.1, DQ467925.1, KM973203.1 and KC691666.1 respectively.
The primer pair for amplifying the beta-tubulin region of the Aspergillus niger genome is as follows:
upstream primer P1: 5'-ACGTATACAACTGCCATTGGAC-3', respectively;
the downstream primer P1: 5'-CACCTCGTTGAAGTAGACGTT-3' are provided.
The primer pair for amplifying the alpha-amylase region of the aspergillus flavus genome is as follows:
upstream primer P2: 5'-ATTCTACAACTTGGCTGATCG-3', respectively;
the downstream primer P2: 5'-TAGTTCAGTACGCCGTCCA-3' are provided.
The primer pair for amplifying the alpha-amylase region of the aspergillus peaking genome is as follows:
upstream primer P3: 5'-TTGCCCGATCTTGACACCAC-3', respectively;
the downstream primer P3: 5'-TAACTGGTTTTCATCCGGCTT-3' are provided.
The primer pair for amplifying the beta-tubulin region of the penicillium genome is as follows:
upstream primer P4: 5'-CAACCAGGTGAGTACAACGA-3', respectively;
the downstream primer P4: 5'-GGACTGACCGAAGACGAAG-3' are provided.
The primer pair for amplifying the RPB2 region of the fusarium genome is as follows:
upstream primer P5: 5'-GTATGGAAGTCGTGGAAGAGTA-3', respectively;
the downstream primer P5: 5'-TTGCATTCGGTAGAGGTCA-3' are provided.
The invention also provides application of the primer combination for detecting the mould in the nuts, which is used for detecting whether toxin-producing fungi exist in the nuts or whether the type of the toxin-producing fungi in the nuts is any one or more of aspergillus niger, aspergillus flavus, aspergillus aggregatus, penicillium and fusarium.
The invention also provides a kit for detecting mildew of nuts, which comprises the primer combination for detecting mildew in nuts.
The invention also provides a method for detecting toxin-producing fungal species in nuts by using the primer combination for detecting mold in nuts, which comprises the following steps:
(1) crushing nuts, placing the crushed nuts into sterile water, and shaking, and carrying out inoculation culture on a shaking solution;
(2) extracting the genome DNA of the cultured hyphae;
(3) performing PCR amplification by taking each primer pair in the primer combination as an upstream primer and a downstream primer respectively and taking genome DNA as a template to obtain an amplification product;
(4) and judging whether the nuts are infected by aspergillus niger, aspergillus flavus, aspergillus aggregatus, penicillium or fusarium according to the molecular weight of the specific strip of the amplification product.
In the step (3), the reaction system of the PCR amplification is as follows: 5 μ L of 10 XMg2+The PCR buffer solution, 4 mu L dNTP, 0.4 mu L Taq DNA polymerase, 1 mu L upstream primer, 1 mu L downstream primer and 5 mu L DNA template are added with sterile ultrapure water to 50 mu L; the reaction conditions of the PCR amplification are as follows: after denaturation at 94 ℃ for 5min, the cycle was repeated at 94 ℃ for 1min, 57 ℃ for 45s and 72 ℃ for 45s for 30 cycles.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the gene sequences of various bacteria are compared in the gene sequence library of NCBI, and specific primers designed at the conservation sites can be used for specifically amplifying common mycotoxin-producing fungi in nuts;
(2) common mould in the nuts can be comprehensively detected by designing primers of common aspergillus, penicillium and fusarium in the nuts;
(3) the result can be judged after DNA extraction, PCR amplification and agarose gel electrophoresis are carried out on the corresponding tissues, the operation is simple, convenient and quick, the whole detection process can be completed within hours generally, and the detection is simple, convenient, quick and efficient.
Drawings
FIG. 1 is a diagram showing the results of PCR amplification when the DNA template is F.oxysporum genome and the upstream and downstream primers are P5; in the figure, lane 1 is a negative control, lanes 2-4 are Fusarium oxysporum, and M is 10kbp Ladder marker;
FIG. 2 is a diagram showing the PCR amplification results when the DNA templates are genomes of Aspergillus niger, Aspergillus flavus, Aspergillus versicolor and Penicillium viridis, respectively, and the upstream and downstream primers are P5; in the figure: lanes 1-2 for Aspergillus niger, lanes 3-4 for Aspergillus flavus, lanes 5-6 for Aspergillus awamori, lanes 7-8 for Penicillium viridis, M is 10kbp Ladder marker;
FIG. 3 is a diagram showing the PCR amplification results when the DNA templates are the genomes of Aspergillus niger, Aspergillus flavus and Aspergillus awamori, and the upstream and downstream primers are P1, respectively; in the figure, lane 1 is a negative control, lanes 2-3 are Aspergillus niger, lanes 4-6 are Aspergillus flavus, lanes 7-9 are Aspergillus awamori, M is 10kbp Ladder marker;
FIG. 4 is a diagram showing the result of PCR amplification when the DNA template is a genome of Penicillium viridis; in the figure, the DNA templates in lanes 1-9 are all the P.viridans genome, but the primers for upstream and downstream in each lane are primer P1 for Aspergillus niger in lanes 1-3, primer P2 for Aspergillus flavus in lanes 4-6, primer P3 for Aspergillus awamori in lanes 7-9, and primer M of 10kbp Ladder marker, respectively;
FIG. 5 is a graph showing the results of PCR amplification when a DNA template is a genome of Fusarium oxysporum; in the figure, the DNA templates in lanes 1-6 are all Fusarium oxysporum, but the primers in the upstream and downstream of each lane are primer P1 for Aspergillus niger in lanes 1-2, primer P3 for Aspergillus awamori genome in lanes 3-4, primer P4 for Penicillium viridis in lanes 5-6, and primer M is 10kbp Ladder marker;
FIG. 6 is a diagram showing the results of PCR amplification when the DNA templates are the genomes of Aspergillus niger, Aspergillus flavus, Aspergillus awamori, Penicillium viridis, and Fusarium oxysporum, respectively, and the upstream and downstream primers are P2; in the figure, lanes 1-2 are Aspergillus niger, lanes 3-4 are Aspergillus flavus, lanes 5-6 are Aspergillus awamori, lanes 7-8 are Penicillium viridis, lanes 9-10 are Fusarium oxysporum, and M is 10kbp Ladder marker;
FIG. 7 is a graph showing the results of PCR amplification when the DNA template is Aspergillus awamori genome and the upstream and downstream primers are P3; in the figure, lane 1 is a negative control, lanes 2-3 are Aspergillus awamori, M is 10kbp Ladder marker;
FIG. 8 is a graph showing the results of PCR amplification when the DNA templates are the genomes of Aspergillus niger and Aspergillus flavus, respectively, and the upstream and downstream primers are P3; in the figure, lane 1 is Aspergillus niger, lane 2 is Aspergillus flavus, lane 3 is a negative control, M is 10kbp Ladder marker;
FIG. 9 is a diagram showing the result of PCR amplification when the DNA template is Penicillium viridans genome and the upstream and downstream primers are P4; in the figure, lane 1 is a negative control, lanes 2-4 are Penicillium viridans, M is 10kbp Ladder marker;
FIG. 10 is a diagram showing PCR amplification results when the DNA templates are genomes of Aspergillus niger, Aspergillus flavus and Aspergillus awamori, and the upstream and downstream primers are P4, respectively; in the figure, lane 1 is Aspergillus niger, lane 2 is Aspergillus flavus, lane 3 is Aspergillus awamori, M is 10kbp Ladder marker;
FIG. 11 is a graph showing the results of detection of the samples in example 3; in the figure, lane 1 is a negative control, lane 2 is an Aspergillus niger primer P1, lane 3 is an Aspergillus flavus primer P2, lane 4 is an Aspergillus awamori primer P3, lane 5 is a Penicillium primer P4, and lane 6 is a Fusarium primer P5.
Detailed Description
The invention is described in detail with reference to the accompanying drawings and the detailed description.
The primers in the following examples were synthesized by Shanghai Biotechnology engineering services, Inc.;
10X of the examples contain Mg2+The PCR buffer of (A) was purchased from Shanghai Bioengineering services, Inc.
dNTPs were purchased from Shanghai Biotechnology engineering services, Inc.
Taq DNA polymerase was purchased from Shanghai Biotechnology engineering services, Inc.;
aspergillus niger (Aspergillus niger CICC 2487), Aspergillus flavus (Aspergillus flavus CICC 2219), Penicillium viridis (Penicillium viridis CICC 4029) and Fusarium oxysporum (Fusarium oxysporum CICC 41029) used in the present invention are all purchased from China Industrial microorganism culture collection management center, and Aspergillus versicolor (Aspergillus nomius ACCC 32558) is purchased from China agricultural microorganism culture collection management center.
Example 1 design and verification of primers
(1) Designing a specific primer:
the BenA (beta-tubulin) region of the Aspergillus niger genome, the Amy-1 (alpha-amylase) region of the Aspergillus flavus genome, the Amy-1 (alpha-amylase) region of the Aspergillus oryzae genome, the BenA (beta-tubulin) region of the Penicillium and the RPB2 region of the Fusarium genome are conserved sequences, strain-specific sequences are downloaded from NCBI database, primers are designed by Oligo7 and Primer5, synthesized by Shanghai Bioengineering technology service, Inc., and Table 1 shows the designed Primer sequences and parameters:
TABLE 1 nut mold primer sequences
(2) The kit method is adopted to extract the genome DNA of the test strain, and the specific method is as follows:
taking hyphae of fusarium oxysporum in a liquid culture medium, and grinding and crushing fungal tissues in liquefied nitrogen; taking not more than 50mg of ground fungal tissue, and placing into a 1.5ml microcentrifuge tube; adding 400 μ L LE Buffer and 4 μ L20 mg/ml RNase A, and mixing well; carrying out warm bath at 65 ℃ for 30min, and carrying out vortex oscillation on the centrifuge tube every 10 min; adding 130 μ L DA Buffer, mixing well, and placing in ice bath for 5 min; centrifuging at 14000Xg for 3 min; transferring the supernatant to a new 1.5ml centrifuge tube; adding 750 mu l 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 tube; adding 500 mu l of G Binding Buffer into Spin column, centrifuging at 10000xg for 30s, and discarding the liquid in the liquid receiving tube; adding 600 mu l of Wash Buffer into Spin column, centrifuging for 30s at 10000xg, discarding the liquid in the liquid receiving tube and repeating the step once; again centrifuging Spin column at 10000xg for 1min and transferring Spin column to a new 1.5ml centrifuge tube; add 100. mu.l to 200. mu.L of Elution Buffer to the item Spin column and incubate for 1min at room temperature; centrifuge at 12000Xg for 1min, discard Spin column, 1.5ml centrifuge tube containing DNA, store at-20 ℃.
Replacing the fusarium oxysporum in the above steps with aspergillus niger, aspergillus flavus, aspergillus aggregatus and fresh penicillium viridis respectively, obtaining the DNA of the aspergillus niger, the aspergillus flavus, the aspergillus aggregatus and the fresh penicillium viridis in the same way, and storing at-20 ℃. Taking the extracted whole genome as a template, and measuring the concentration and purity of the extracted DNA by using a Nanodrop, wherein the concentration of the obtained Aspergillus niger genome is 7.2 ng/mu l, and A260/A280 is 2.08; the concentration of the aspergillus flavus is 7.2 ng/mu l, and the A260/A280 is 2.03; the genome concentration of Aspergillus awamori is 6.9 ng/mul, and the A260/A280 is 1.91; the genome concentration of the green penicillium is 3.3 ng/mu l, and A260/A280 is 1.70; the genome concentration of Fusarium oxysporum was 14 ng/. mu.l, and A260/A280 was 1.84.
(3) And (3) verifying the primers:
the PCR reaction system is as follows: 5 μ L of 10 XMg2+The PCR buffer solution of (1) above, 4. mu.L dNTP, 0.4. mu.L Taq DNA polymerase, 1. mu.L each of the upstream and downstream primers, and 5. mu.L DNA template, and sterile ultrapure water was added to 50. mu.L.
The amplification procedure was: after denaturation at 94 ℃ for 5min, circulation is carried out, wherein the temperature is 94 ℃ for 1min, 57 ℃ for 45s and 72 ℃ for 45s, 30 cycles are carried out, and the temperature is 72 ℃ for 10 min.
And (3) separating 2.5 mu L of PCR product by using 1% agarose electrophoresis, observing the length of the specific strip under an ultraviolet lamp after GelRed staining, wherein the lengths of the specific strips of Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Penicillium viridis and Fusarium oxysporum are respectively 300bp, 197bp, 360bp, 202bp and 467 bp.
In actual detection, taking the genome DNA of an object to be detected as a template, respectively carrying out PCR amplification by using the primers, and if an amplification product with a corresponding length can be specifically amplified, judging that corresponding fungi exist in the object to be detected; otherwise the corresponding fungus is absent. For example, if the length of the band of the product amplified by using the upstream primer P1 and the downstream primer P1 is 300bp, the existence of Aspergillus niger in the analyte is indicated; if the length of the band of the product amplified by using the upstream primer P2 and the downstream primer P2 is 197bp, the Aspergillus flavus still exists in the substance to be detected.
Example 2 specificity test of each primer pair
(1) Extraction of genomic DNA from Fusarium oxysporum
The genomes of Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Penicillium viridissimum, and Fusarium oxysporum were each extracted as described in step (2) of example 1. The concentration of the obtained Aspergillus niger genome is 7.2 ng/mu l, and the A260/A280 is 2.08; the concentration of the aspergillus flavus is 7.2 ng/mu l, and the A260/A280 is 2.03; the genome concentration of Aspergillus awamori is 6.9 ng/mul, and the A260/A280 is 1.91; the genome concentration of the green penicillium is 3.3 ng/mu l, and A260/A280 is 1.70; the genome concentration of Fusarium oxysporum was 14 ng/. mu.l, and A260/A280 was 1.84.
(2) Establishment of PCR System
The PCR reaction system is as follows: 5 μ L of 10 XMg2+The PCR buffer solution of (3), 4. mu.L dNTP, 0.4. mu.L Taq DNA polymerase, 1. mu.L each of the upstream and downstream primers, and 5. mu.L DNA template, and sterile ultrapure water was added to 50. mu.L.
The amplification procedure was: after denaturation at 94 ℃ for 5min, circulation is carried out, wherein the temperature is 94 ℃ for 1min, 57 ℃ for 45s and 72 ℃ for 45s, 30 cycles are carried out, and the temperature is 72 ℃ for 10 min.
When the DNA template is a fusarium oxysporum genome, the upstream primer P5 and the downstream primer P5 are used as the upstream primer and the downstream primer, a specific strip with the molecular weight of 467bp appears in an amplification product, and the amplification result is shown in figure 1, which indicates that the amplified target product is a target gene of fusarium oxysporum. If the genome of the fusarium oxysporum is replaced by the genome of aspergillus niger, the genome of aspergillus flavus, the genome of aspergillus awamori and the genome of penicillium viridans respectively, and the upstream primer and the downstream primer are also the upstream primer and the downstream primer P5, products with any specific bands cannot be amplified, and the amplification result is shown in figure 2, which shows that the primers have strong specificity and can be used for quickly and reliably detecting whether the nuts are infected by the fusarium oxysporum in production practice.
When the DNA template is an Aspergillus niger genome, the upstream primer P1 and the downstream primer P1 are used as the upstream primer and the downstream primer, a specific band with the molecular weight of 300bp appears in an amplification product, and the amplified target product is the target gene of Aspergillus niger. If the Aspergillus niger genome is replaced by the Aspergillus flavus genome, the Aspergillus awamori genome, the Penicillium viridis genome and the Fusarium oxysporum genome respectively, the upstream primer and the downstream primer are also the upstream primer and the downstream primer P1, and the amplification results are shown in FIGS. 3-5. This shows that the primer has strong specificity and can be used for rapid and reliable detection in production practice and whether the nuts are infected by Aspergillus niger.
When the DNA template is an Aspergillus flavus genome, the upstream primer P2 and the downstream primer P2 are used as the upstream primer and the downstream primer, a specific band with molecular weight of 197bp appears in an amplified product, and the amplified target product is the target gene of the Aspergillus flavus. If the Aspergillus flavus genome is replaced by the Aspergillus niger genome, the Aspergillus awamori genome, the Penicillium viridis genome and the Fusarium oxysporum genome respectively, and the upstream primer and the downstream primer are also the upstream primer and the downstream primer P2, no specific strip product can be amplified, and the amplification result is shown in FIG. 6. This shows that the primer has strong specificity, and can be used for quickly and reliably detecting whether the nuts are infected by aspergillus flavus in production practice.
When the DNA template is Aspergillus awamori genome, the upstream primer P3 and the downstream primer P3 are used as the upstream primer and the downstream primer, a specific band with the molecular weight of 360bp appears in the amplification product, and the amplification result is shown in FIG. 7, which indicates that the amplified target product is Aspergillus awamori target gene. If the Aspergillus awamori genome is replaced by the Aspergillus niger genome, the Aspergillus flavus genome, the Penicillium viridis genome and the Fusarium oxysporum genome respectively, and the upstream primer and the downstream primer are also the upstream primer and the downstream primer P3, the amplification result is shown in FIGS. 4-5 and 8, which shows that the primers have strong specificity and can be used for quickly and reliably detecting whether the nuts are infected with the Aspergillus awamori in production practice.
When the DNA template is a genome of Penicillium viridescens, the upstream primer P4 and the downstream primer P4 are used as the upstream primer and the downstream primer, a specific band with a molecular weight of 202bp appears in the amplification product, and the amplification result is shown in FIG. 9, which indicates that the target product obtained by amplification is the target gene of Penicillium viridescens. If the genome of the fresh penicillium viridans is replaced by the genome of aspergillus niger, the genome of aspergillus flavus, the genome of aspergillus awamori and the genome of fusarium oxysporum respectively, and the upstream primer and the downstream primer are also the upstream primer and the downstream primer P4, products with any specific bands cannot be amplified, and the amplification results are shown in fig. 5 and fig. 10, which shows that the primers have strong specificity and can be used for quickly and reliably detecting whether the nuts are infected with the fresh penicillium viridans in production practice.
EXAMPLE 3 detection of naturally contaminated samples
(1) Sample treatment:
sampling from a Lingan industrial park in Lingan city of Hangzhou, Zhejiang, taking 25g of nuts, crushing, adding 225mL of sterilized water, oscillating for 10min, adding 200 mu L of oscillated solution into 100mL of potato liquid culture medium, and culturing at 28 ℃ for 40 h.
(2) Sample DNA extraction
The mycelia in the medium of step (1) were collected, and genomic DNA was sampled according to the method of step (2) of example 1.
(3) Nut sample PCR system establishment
And (3) PCR reaction system: 5 μ L of 10 XMg2+The PCR buffer solution of (1) above, 4. mu.L dNTP, 0.4. mu.L Taq DNA polymerase, 1. mu.L each of the upstream primer and the downstream primer, 5. mu.L of the sample DNA template extracted in step (2), and 33.6. mu.L of sterile ultrapure water.
The amplification procedure was: after denaturation at 94 ℃ for 5min, the cycle was repeated at 94 ℃ for 1min, 57 ℃ for 45s and 72 ℃ for 45s for 30 cycles.
PCR amplification was performed using the five primer sets of example 1, respectively, and 2.5. mu.L of the PCR product was electrophoresed on 1% agarose gel, stained with GelRed, and the results were judged according to the band size under an ultraviolet lamp.
The detection result is shown in FIG. 11, and the amplification product has a specific band with molecular weight of 197bp, so that the nut infected with Aspergillus flavus can be judged.
The above detailed description of a primer combination for the mildew detection of nuts, a kit and a method for detecting mycotoxin-producing fungal species in nuts with reference to the embodiments is illustrative and not restrictive, and several embodiments can be cited within the scope of the present invention, so that variations and modifications thereof without departing from the general concept of the present invention shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110> three squirrels (Nanjing) food technology research and development Co., Ltd, Jiangnan university of three squirrels
<120> primer combination and kit for detecting mould in nuts and detection method for mycotoxin-producing fungus species in nuts
<130> 1
<160> 10
<170> PatentIn version 3.3
<210> 1
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<212> DNA
<213> upstream primer P1
<400> 1
5'-acgtatacaa ctgccattgg ac-3' 22
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<212> DNA
<213> downstream primer P1
<400> 2
5'-cacctcgttg aagtagacgt t-3' 21
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<212> DNA
<213> upstream primer P2
<400> 3
5'-attctacaac ttggctgatc g-3' 21
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<213> downstream primer P2
<400> 4
5'-tagttcagta cgccgtcca-3' 19
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<213> upstream primer P3
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5'-ttgcccgatc ttgacaccac-3' 20
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<213> downstream primer P3
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5'-taactggttt tcatccggct t-3' 21
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<212> DNA
<213> upstream primer P4
<400> 7
5'-caaccaggtg agtacaacga-3' 20
<210> 8
<211> 19
<212> DNA
<213> downstream primer P4
<400> 8
5'-ggactgaccg aagacgaag-3' 19
<210> 9
<211> 22
<212> DNA
<213> upstream primer P5
<400> 9
5'-gtatggaagt cgtggaagag ta-3' 22
<210> 10
<211> 19
<212> DNA
<213> downstream primer P5
<400> 10
5'-ttgcattcgg tagaggtca-3' 19
Claims (10)
1. A primer combination for detecting mold in nuts, the primer combination comprising:
amplifying a primer pair of a beta-tubulin region of an Aspergillus niger genome;
amplifying a primer pair of an alpha-amylase region of an aspergillus flavus genome;
amplifying a primer pair of an alpha-amylase region of an aspergillus peaking genome;
amplifying a primer pair of a beta-tubulin region of the penicillium genome;
primer pairs for amplifying the RPB2 region of fusarium genome.
2. The primer combination for detecting mold in nuts as claimed in claim 1, wherein said primer pair for amplifying β -tubulin region of the A.niger genome is:
upstream primer P1: 5'-ACGTATACAACTGCCATTGGAC-3', respectively;
the downstream primer P1: 5'-CACCTCGTTGAAGTAGACGTT-3' are provided.
3. The primer combination for detecting mold in nuts as claimed in claim 1, wherein the primer pair for amplifying the alpha-amylase region of the genome of Aspergillus flavus is:
upstream primer P2: 5'-ATTCTACAACTTGGCTGATCG-3', respectively;
the downstream primer P2: 5'-TAGTTCAGTACGCCGTCCA-3' are provided.
4. The primer combination for detecting mold in nuts as claimed in claim 1, wherein said primer pair for amplifying the α -amylase region of the aspergillus peaking genome is:
upstream primer P3: 5'-TTGCCCGATCTTGACACCAC-3', respectively;
the downstream primer P3: 5'-TAACTGGTTTTCATCCGGCTT-3' are provided.
5. The primer combination for detecting molds in nuts as claimed in claim 1, wherein said primer pair for amplifying β -tubulin region of penicillium genome is:
upstream primer P4: 5'-CAACCAGGTGAGTACAACGA-3', respectively;
the downstream primer P4: 5'-GGACTGACCGAAGACGAAG-3' are provided.
6. The primer combination for detecting mold in nuts as claimed in claim 1, wherein said primer pair for amplifying RPB2 region of fusarium genome is:
upstream primer P5: 5'-GTATGGAAGTCGTGGAAGAGTA-3', respectively;
the downstream primer P5: 5'-TTGCATTCGGTAGAGGTCA-3' are provided.
7. Use of the primer combination according to any one of claims 1-6 for the detection of mould in nuts, wherein the primer combination is used for detecting the presence of mycotoxin-producing fungi in nuts, or for detecting the presence of any one or more of Aspergillus niger, Aspergillus flavus, Aspergillus awamori, Penicillium sp.
8. A kit for the detection of mildew in nuts, said kit comprising the primer combination for the detection of mildew in nuts according to any one of claims 1 to 6.
9. A method for detecting mycotoxin-producing fungal species in nuts using the primer combination for detecting mold in nuts of any one of claims 1-6, comprising the steps of:
(1) crushing nuts, placing the crushed nuts into sterile water, and shaking, and carrying out inoculation culture on a shaking solution;
(2) extracting the genome DNA of the cultured hyphae;
(3) performing PCR amplification by taking each primer pair in the primer combination as an upstream primer and a downstream primer respectively and taking genome DNA as a template to obtain an amplification product;
(4) and judging whether the nuts are infected by aspergillus niger, aspergillus flavus, aspergillus aggregatus, penicillium or fusarium according to the molecular weight of the specific strip of the amplification product.
10. The method of claim 9, wherein in the step (3), the reaction system of the PCR amplification is: 5 μ L of 10 XMg2+The PCR buffer solution, 4 mu L dNTP, 0.4 mu L Taq DNA polymerase, 1 mu L upstream primer, 1 mu L downstream primer and 5 mu L DNA template are added with sterile ultrapure water to 50 mu L; the reaction conditions of the PCR amplification are as follows: after denaturation is carried out for 5min at the temperature of 94 ℃, circulation is carried out,94 ℃ for 1min, 57 ℃ for 45s and 72 ℃ for 45s, for 30 cycles.
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| US20140141993A1 (en) * | 2011-06-09 | 2014-05-22 | Toyo Seikan Group Holdings, Ltd. | Method for detecting fungi, reaction solution for pcr, and carrier for detecting fungi |
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| CN113293225A (en) * | 2021-06-29 | 2021-08-24 | 江南大学 | Primer probe combination capable of specifically recognizing aspergillus, penicillium and fusarium and application thereof |
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2021
- 2021-09-09 CN CN202111055528.5A patent/CN113684303A/en active Pending
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|---|---|---|---|---|
| US20080014582A1 (en) * | 2006-04-01 | 2008-01-17 | Hooper Dennis G | Methods and compositions for detecting fungi and mycotoxins |
| US20140141993A1 (en) * | 2011-06-09 | 2014-05-22 | Toyo Seikan Group Holdings, Ltd. | Method for detecting fungi, reaction solution for pcr, and carrier for detecting fungi |
| CN105648038A (en) * | 2014-08-21 | 2016-06-08 | 黄耀江 | LAMP (loop-mediated isothermal amplification) universal primer for detecting aspergillus toxin-producing fungi and kit containing primer |
| CN113293225A (en) * | 2021-06-29 | 2021-08-24 | 江南大学 | Primer probe combination capable of specifically recognizing aspergillus, penicillium and fusarium and application thereof |
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