Fluorescent Quantitative PCR Detection Method for Spore Concentration of Aflatoxin-Producing Aspergillus flavus Strains

Abstract

The present invention discloses a fluorescence quantitative PCR detection method for the spore concentration of Aspergillus flavus toxin-producing strains. In this method, the fluorescence quantitative PCR primer set for detecting the spore concentration of Aspergillus flavus toxin-producing strains is composed of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing. The detection method established in this study is simple, rapid, highly specific, stable, and sensitive, and is suitable for large-scale detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for fluorescence quantitative PCR detection of spore concentration of aflatoxin-producing strains of Aspergillus flavus. Background Art

[0002] Aflatoxins (AFT) are mainly secondary metabolites produced by fungi Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius. They have the "three carcinogenic effects" of carcinogenesis, teratogenesis, and cell mutagenesis, and are widely present in agricultural products such as peanuts, corn, wheat, and rice, and are also found in macaroni, seasonings, milk, and edible oils, seriously endangering the health of humans, livestock, and poultry. Aflatoxins mainly include B1, B2, G1, G2, M1, M2, GM, P1, Q1, and toxicol, etc. In naturally contaminated samples, B1 is the most contaminated, and B1 has the greatest toxicity and the strongest carcinogenicity. In 1993, aflatoxin was designated as a Class I carcinogen by the cancer research agency of the World Health Organization (WHO). Approximately 25% of the world's annual crops are contaminated by pathogens and their toxins, and approximately 2% of the crops lose their nutritional and economic value due to severe contamination. Therefore, the detection method of aflatoxin has always been the focus of domestic and foreign research. Currently, the conventional detection methods for aflatoxin mainly include immunological detection methods based on antigen-antibody reactions such as colloidal gold detection method, enzyme-linked immunosorbent assay, and fluorescence immunoassay, which are usually applied to the screening of aflatoxin; the accurate quantitative detection methods mainly include instrument detection methods based on high-end detection equipment such as liquid chromatography and liquid chromatography-tandem mass spectrometry. These detection methods are time-consuming, laborious, complex to operate, and costly.

[0003] Aflatoxins in grains and foods are mainly produced by Aspergillus flavus, while Aspergillus parasiticus and Aspergillus nomius are rare. According to research, only 10% of Aspergillus flavus strains in nature can produce aflatoxins. Therefore, the detection of aflatoxin-producing strains is of great significance for early warning of aflatoxin pollution risk, and at the same time can save the detection cost of aflatoxin. However, there are currently few methods for detecting aflatoxin-producing strains. It is reported that the aflO, aflD, and aflP genes are key genes in the synthesis process of aflatoxin B1. The strains expressing these three genes are all aflatoxin-producing strains detected by HPLC. Therefore, it is possible to infer whether the tested strain is an aflatoxin-producing strain or its potential for producing toxins by detecting the expression of these three genes, so as to quickly and accurately judge the potential aflatoxin pollution risk in the tested sample. Summary of the Invention

[0004] Based on this, in this study, the key gene aflD for aflatoxin production by Aspergillus flavus was used as the detection target, and specific primers were designed; and directly using the total DNA extracted from Aspergillus flavus spores as a template, the real-time fluorescence quantitative PCR technology was used to detect the aflatoxin-producing strains of Aspergillus flavus in peanuts, and a relationship model was established between the fluorescence quantitative Ct value of the sample and the spore concentration of Aspergillus flavus strains to infer the potential contamination risk of aflatoxin.

[0005] The present invention provides a fluorescence quantitative PCR primer set for detecting the spore concentration of aflatoxin-producing strains of Aspergillus flavus, which is composed of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing.

[0006] The present invention also provides a fluorescence quantitative PCR kit for detecting the spore concentration of aflatoxin-producing strains of Aspergillus flavus, including the fluorescence quantitative PCR primer set described in Claim 1.

[0007] The kit also includes PCR reaction reagents.

[0008] The application of the above fluorescence quantitative PCR primer set and the above kit in detecting the spore concentration of aflatoxin-producing strains of Aspergillus flavus also belongs to the protection scope of the present invention.

[0009] The fluorescence quantitative PCR method provided by the present invention for detecting the spore concentration of aflatoxin-producing strains of Aspergillus flavus includes the following steps:

[0010] 1) Suspending the sample to be tested in water to obtain a spore suspension;

[0011] 2) Extracting the DNA of the sample to be tested;

[0012] 3) Using the DNA of the sample to be tested as a template, performing fluorescence quantitative PCR detection with the fluorescence quantitative PCR primer set described in Claim 1 to obtain a Ct value;

[0013] 4) Substituting the Ct value obtained in step 3) into the linear curve y = -2.7406x + 48.722 of the spore concentration of aflatoxin-producing strains of Aspergillus flavus and the Ct value obtained in step 1), where y is the fluorescence quantitative PCR Ct value, x = lgA, where A is the spore concentration of aflatoxin-producing strains of Aspergillus flavus, with the unit of number / ml, to convert and obtain the spore concentration of aflatoxin-producing strains of Aspergillus flavus in the spore suspension of the sample to be tested.

[0014] Among them, the reaction system of the fluorescence quantitative PCR is a 20 μL system, including 1 μL of DNA template, 0.4 μL of the primer shown in Sequence 1 in the sequence listing (10 μM), 0.4 μL of the primer shown in Sequence 2 in the sequence listing (10 μM), 10 μL of Mix, and 8.2 μL of ddH2O.

[0015] The reaction procedure of the fluorescence quantitative PCR is as follows: first, at 95°C for 2 min, then at 95°C for 10 s, at 62.5°C for 30 s, for 40 cycles, and finally at 95°C for 15 s, at 60°C for 1 min, at 95°C for 1 s.

[0016] In the said step 2, the method for extracting DNA of the sample to be detected is as follows: the spore suspension of the sample to be detected is placed in a 1.5 mL centrifuge tube, centrifuged at 12000 rpm in a centrifuge for 15 min, and then the supernatant is removed. The spore precipitate is frozen with liquid nitrogen, ground, 200 μL of ddH2O is added to the centrifuge tube, and it is placed in boiling water for 9 min, centrifuged at 14000 r, at 4°C for 15 min, and the obtained supernatant is the Aspergillus flavus spore DNA solution.

[0017] In step 1), the method for obtaining the spore suspension of the sample to be detected is to mix the sample with water and shake well to obtain the spore suspension of the Aspergillus flavus toxin-producing strain.

[0018] In the said step 1), it also includes transferring the obtained spore suspension of the Aspergillus flavus toxin-producing strain to a 50 mL centrifuge tube and shaking well for 3 min; filtering the spore suspension in the centrifuge tube through sterilized absorbent cotton to remove other substances such as the culture medium, then rinsing the absorbent cotton with 5 mL of ddH2O / RNase-free water to obtain a pure spore suspension, centrifuging at 12000 rpm in a centrifuge for 15 min, removing the supernatant, and adding 1 mL of ddH2O / RNase-free water to concentrate the spore suspension.

[0019] The method of the present invention is simple, rapid, highly specific, stable, and sensitive, and is suitable for large-scale detection. Description of the Drawings

[0020] Figure 1 is the front side of the FPA medium and the back side of the AFPA medium

[0021] Figure 2 is the linear relationship diagram between the spore concentration of Aspergillus flavus and the DNA concentration.

[0022] Figure 3 is the electrophoresis diagram of the PCR amplification product of the specific primer aflD gene; M: DL1000 DNA Marker; 1: the template is ddH2O; 2-3: the PCR amplification product of the aflD gene.

[0023] Figure 4 is the standard curve diagram of the logarithm of the spore concentration of the Aspergillus flavus toxin-producing strain - Ct value

[0024] Figure 5 is the standard curve diagram of the logarithm of the spore DNA concentration of the Aspergillus flavus toxin-producing strain - Ct value.

[0025] Figure 6 : Standard curve graph of nor-1 gene copy number - Ct value in the literature

[0026] Figure 7 : Gel running comparison graph of primer products in the literature and primer products of this method

[0027] (M: DL1000 DNA Marker; CK: Template is ddH2O; 1 - 2: PCR amplification product of nor gene; 3 - 4: PCR amplification product of aflD gene)

[0028] Figure 8 . Curve relationship graph of percentage absorbance value of aflatoxin standard at 450 nm Detailed implementation mode

[0029] The present invention will be further described below with reference to the attached drawings and in combination with specific embodiments for better understanding. For those not specified in the embodiments in terms of specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase

[0030] Example 1: Fluorescent quantitative PCR method for detecting spores of aflatoxin-producing strains of Aspergillus flavus

[0031] 1. Materials

[0032] 1.1 Test strains

[0033] Strain: Aspergilus flavus (BNCC142803), purchased from Wuhan Jitai Biotechnology Co., Ltd

[0034] 1.2 Reagents and instruments

[0035] Table 1. Reagents and instruments

[0036]

[0037]

[0038] 2 Test methods

[0039] 2.1 Strain activation, culture and toxin production identification

[0040] Before the formal experiment starts, after activating 1 - 2 generations according to the activation steps and requirements in the instruction manual of Aspergillus flavus freeze-dried powder of BNCC 142803, take the germinated spores, wash the spores with sterile normal saline and concentrate to a final concentration of 10 6 cells / mL, inoculate on PDA medium, and culture at 28 °C under aerobic and light-free conditions for 5 - 7 days

[0041] To determine that the Aspergillus flavus strain purchased from Wuhan Jitai Biotechnology Co., Ltd. is indeed a toxin-producing strain and is not contaminated by Aspergillus parasiticus, the AFPA medium was used to verify the strain. As Figure 1 shown, it is a toxin-producing strain.

[0042] The BNCC 142803 strain produces aflatoxins B1, B2, G1, and G2. During the growth of Aspergillus flavus, an acidic substance is produced, which reacts with the metal iron ions in the AFPA medium, and an orange-yellow color appears at the center of the reverse side of the colony. If the center of the reverse side of the colony is white, it is contaminated by Aspergillus parasiticus. From Figure 1 it can be seen that this strain is not contaminated by Aspergillus parasiticus.

[0043] 2.2 Preparation of Spore Suspension of Aspergillus flavus Toxin-producing Strain

[0044] Take 5 mL of ddH2O / RNase-free water and place it on the PDA medium covered with the Aspergillus flavus toxin-producing strain. Gently scrape the surface of the PDA medium with an inoculation loop to obtain the spore suspension of the Aspergillus flavus toxin-producing strain and transfer it to a 50 mL centrifuge tube and shake it evenly for 3 min. Filter the evenly mixed spore suspension in the centrifuge tube with sterilized absorbent cotton to remove other substances such as the medium. Then rinse the absorbent cotton with 5 mL of ddH2O / RNase-free water to obtain a pure spore suspension. After centrifuging at 12,000 rpm for 15 min in a centrifuge, discard the supernatant and add 1 mL of ddH2O / RNase-free water to concentrate the spore suspension. Count the obtained spore suspension with a hemocytometer to obtain the concentration of this spore suspension.

[0045] 2.3 DNA Extraction and Concentration Determination of Spore Suspension of Aspergillus flavus Toxin-producing Strain

[0046] The spore suspension of Aspergillus flavus with a spore concentration of 1.55×10 8 per mL was diluted in a five-fold gradient. The obtained suspension concentrations were 3.10×10 7 , 6.20×10 6 , 1.24×10 6 , 2.48×10 5 and 4.96×10 4 / mL, and set up a blank control at the same time. Take 200μL of spore suspension of each concentration into a 1.5mL centrifuge tube, centrifuge at 12000rpm for 15min in a centrifuge, and remove the supernatant. The spore precipitate was frozen with liquid nitrogen, ground, added with 200μLddH2O in a centrifuge tube, placed in boiling water for 9min, 14000r, 4℃, 15min, and the resulting supernatant was the DNA solution of Aspergillus flavus spores. The DNA concentration was determined using a multifunctional microplate reader (InfiniteM200 PRO) and an ultra-micro spectrophotometer (DeNovix), and the experiment was repeated at least 3 times.

[0047] As shown in Table 2, the spore suspension of Aspergillus flavus toxin-producing strains with high quality can be obtained by filtering with absorbent cotton. The total DNA of Aspergillus flavus with high purity and quality can be obtained by using the spore suspension, freezing with liquid nitrogen, grinding and heating centrifugation, and its OD260 / 280 is in the range of 1.70-1.93. And there is a good linear relationship between the concentration of Aspergillus flavus spores and the concentration of DNA, y=5E-06x-0.5722 (y is the DNA concentration, in ng / μL, x is the concentration of Aspergillus flavus spores, in units / ml, Figure 2 ), correlation coefficient R 2 The value reached 0.9999. This shows that the method for obtaining aflatoxin spores and extracting DNA established in this study is reliable, easy to operate and economical.

[0048] Table 2. DNA concentration and quality test table of Aspergillus flavus spore extraction

[0049]

[0050] 2.4 Specific primer design

[0051] The key gene aflD in the synthesis of aflatoxin B1 was used as the detection target. Specific primers were designed using Primer5.0 software and synthesized by Beijing Liuhe BGI Technology Co., Ltd. The estimated product length is about 200 bp. The primer sequences are as follows:

[0052] aflDF: 5'-CCAGACATTGCGGGAGGAG-3' (sequence 1 in the sequence list)

[0053] aflDR: 5'-GCAGCATCAGGCGGGTT-3' (sequence 2 in the sequence list)

[0054] 2.5 PCR amplification and identification of aflD gene of aflatoxin-producing strains

[0055] Primers were designed and synthesized using 2.4, and the aflD gene was amplified by PCR. The PCR products were recovered and sequenced (Beijing Liuhe Huada Gene Technology Co., Ltd.), and the gene sequences were identified by alignment with the NCBI database. The PCR reaction system was as follows: a 50 μL system, including 1 μL of template, 1 μL of Taq enzyme, 1 μL of 3F, 1 μL of 3R, 5 μL of PCR Buffer, 4 μL of dNTP, and 37 μL of ddH2O. The reaction conditions were as follows: 95°C for 5 min, (95°C, 30 s, 62.5°C for 30 s, 72°C for 1 min for 36 cycles), 72°C for 10 min, 4°C, and the experiment was repeated at least 3 times.

[0056] As Figure 3 shown, the specific primers designed in this study amplified clear target bands without non-specific bands. The sequence determination and splicing results of the PCR amplification products were as shown in Sequence 3 in the sequence list. After aligning the recovered and sequenced results of the amplification products with the NCBI database, the amplified gene was determined to be the key gene aflD fragment for aflatoxin B1 production, and its length was 172 bp (Sequence 3 in the sequence list).

[0057] 2.6 Establishment of an RT-PCR detection model for the spore concentration of aflatoxin-producing Aspergillus flavus strains

[0058] Using the aflD gene as the target, the DNA samples of Aspergillus flavus obtained in 2.3 were detected by fluorescence quantitative PCR, and the relationship between the Ct value of fluorescence quantitative PCR and the spore concentration of Aspergillus flavus was established. The fluorescence quantitative PCR reaction system was as follows: a 20 μL system, including 1 μL of template, 0.4 μL of forward primer aflDF (10 μM), 0.4 μL of reverse primer aflDR (10 μM), 10 μL of Mix, and 8.2 μL of ddH2O. The reaction conditions were as follows: first 95°C for 2 min, then 95°C, 10 s, 62.5°C for 30 s for 40 cycles, and finally 95°C for 15 s, 60°C for 1 min, 95°C for 1 s. The experiment was repeated at least three times.

[0059] As shown in Table 3, as the spore concentration of aflatoxin-producing Aspergillus flavus decreased, the DNA concentration decreased and the Ct value of fluorescence quantitative PCR increased. Using the logarithm of the spore concentration of aflatoxin-producing Aspergillus flavus as the abscissa and the Ct value as the ordinate, a linear curve of the lg value (x) of the spore concentration of Aspergillus flavus and the Ct value (y) was established, showing a good correlation y = -2.7406x + 48.722 (y is the Ct value of fluorescence quantitative PCR, x = lgA, where A is the spore concentration of aflatoxin-producing Aspergillus flavus strains, with the unit of cells / ml), and the R 2 value reached 0.9807 ( Figure 4)。It shows that there is a strong linear relationship between the concentration of Aspergillus flavus spores and the expression of the key control gene aflD in the toxin-producing process of the toxin-producing strain, and the spore concentration of the Aspergillus flavus toxin-producing strain can be quantified by detecting the expression of the aflD gene by fluorescence quantitative PCR. At the same time, establish the relationship between the Ct value (y) and the lg value of the spore DNA concentration of the Aspergillus flavus toxin-producing strain (x) as y = -2.825x + 28.08 (y is the fluorescence quantitative PCR Ct value, x = lgA, where A is the spore DNA concentration of the Aspergillus flavus toxin-producing strain, unit: ng / μL), and find that its R 2 value reaches 0.9831( Figure 5 ), indicating that there is also a good correlation between the Ct value and the spore DNA concentration of the Aspergillus flavus toxin-producing strain. That is, two models are established in this study. Using the specific primers screened and designed in this study, the spore concentration and DNA concentration of the Aspergillus flavus toxin-producing strain can be quantitatively determined by fluorescence quantitative PCR technology respectively.

[0060] Table 3. Aspergillus flavus spore concentration and Ct value detected by fluorescence quantitative PCR

[0061]

[0062] To sum up, this study established a fluorescence quantitative PCR detection method for Aspergillus flavus toxin-producing strain spores in agricultural products such as peanuts, corn, wheat, and rice in China. This method is simple, convenient, economical, and fast from sample DNA extraction to detection, and is of great significance for the monitoring and prevention of aflatoxin contamination in agricultural products such as peanuts, corn, wheat, and rice in China.

[0063] Example 2. Application of RT-PCR detection model for Aspergillus flavus toxin-producing strain spore concentration

[0064] For the model established in 2.6 of Example 1, in order to test its accuracy, the purchased peanuts were rinsed several times with sterile water and alcohol, dried after ensuring no impurities, and then inoculated with the Aspergillus flavus toxin-producing strain (BNCC142803). 20 peanuts were placed in each petri dish and cultured under the same culture conditions, and a control inoculated with clear water was set at the same time. After they became moldy, 20 peanuts were placed in a sterile beaker, and spores were extracted with ddH2O. After shaking well, a spore suspension of the Aspergillus flavus toxin-producing strain was obtained and transferred to a 50 mL centrifuge tube and shaken well for 3 min. The spore suspension in the centrifuge tube was filtered through sterilized absorbent cotton to remove other substances such as the culture medium. Then the absorbent cotton was rinsed with 5 mL ddH2O to obtain a pure spore suspension. After centrifuging at 12000 rpm for 15 min in a centrifuge, the supernatant was removed, and 1 mL ddH2O was added to concentrate the spore suspension. The obtained spore suspension was counted with a hemocytometer under a microscope, and DNA was prepared and detected by fluorescence quantitative PCR successively according to the methods of 2.3 and 2.6. The experiment was repeated three times.

[0065] The specific detection method is as follows:

[0066] 1) Prepare DNA: Place the spore suspension in a 1.5 mL centrifuge tube. After centrifuging at 12,000 rpm for 15 min in a centrifuge, discard the supernatant. Freeze the spore precipitate in liquid nitrogen, grind it, add 200 μL of ddH₂O to the centrifuge tube, place it in boiling water for 9 min, centrifuge at 14,000 r, 4 °C for 15 min, and the obtained supernatant is the Aspergillus flavus spore DNA solution.

[0067] 2) Use fluorescence quantitative PCR: Using the Aspergillus flavus spore DNA solution as a template, obtain the Ct value by fluorescence quantitative PCR method. The primer sequences are as follows:

[0068] aflDF: 5’-CCAGACATTGCGGGAGGAG-3’ (Sequence 1 in the sequence listing)

[0069] aflDR: 5’-GCAGCATCAGGCGGGTT-3’ (Sequence 2 in the sequence listing)

[0070] The fluorescence quantitative PCR reaction system is as follows: 20 μL system, including 1 μL of template, 0.4 μL of forward primer aflDF (10 μM), 0.4 μL of reverse primer aflDR (10 μM), 10 μL of Mix, and 8.2 μL of ddH₂O. The reaction conditions are as follows: 95 °C for 2 min, (95 °C, 10 s, 62.5 °C for 30 s, 40 cycles), 95 °C for 15 s, 60 °C for 1 min, 95 °C for 1 s. (Please try to use Chinese for each stage).

[0071] 3) Calculate the spore concentration of Aspergillus flavus toxigenic strains: Substitute the Ct value obtained in step 2) into the formula y = -2.7406x + 48.722, where y is the fluorescence quantitative PCR Ct value, x = lgA, and A is the spore concentration of Aspergillus flavus toxigenic strains, with the unit of number / ml, to obtain the spore concentration of Aspergillus flavus toxigenic strains.

[0072] The results are shown in Table 4. The model established in this study was used to calculate the spore concentration of Aspergillus flavus toxigenic strains in the samples, and the differences between the calculated values and the actual microscopic examination values were compared. The results showed that there was no significant difference between the calculated number of Aspergillus flavus toxigenic strain spores in the test samples and the measured microscopic examination spore concentration of toxigenic strains. The detection method established in this study can accurately quantify the spore concentration of Aspergillus flavus toxigenic strains in peanuts.

[0073] Table 4. Application verification of RT-PCR for detecting the spore concentration of Aspergillus flavus toxigenic strains

[0074]

[0075]

[0076] Note: During actual operation, try to concentrate the spores to a high concentration for more accurate results.

[0077] Comparative Example 1:

[0078] Yan Li et al. (Yan Li, 2012) established a rapid detection method for aflatoxin-producing strains in the article "Research and Application of Detection Methods for Aflatoxin-Producing Fungi". This method also uses the aflD gene as the target gene, and the primer sequences are: nor-1-S: 5'-GTCCAAGCAACAGGCCAAGT-3'; nor-1-R: 5'-TCGTGCATGTTGGTGATGGT-3'. The standard curve established uses the logarithm of the aflD gene copy number as the abscissa and the Ct value as the ordinate. The standard curve is as Figure 6 shown. This detection method can only quantify the aflD gene copy number and cannot quantify aflatoxin-producing strains.

[0079] The primers of this comparative example and the primers of the present invention were used to perform PCR amplification on aflatoxin-producing strains respectively. The amplification results are as Figure 7 shown, where lanes 1 and 2 are the amplification results of the primers of this comparative example, and lanes 3 and 4 are the amplification results of the primers of the present invention. It shows that the amplification products of the primers of the present invention have clearer bands.

[0080] Comparative Example 2:

[0081] The following results were obtained by operating the Aflatoxin ELISA Detection Kit (Beijing Beilo Biotechnology Co., Ltd.) according to the kit operation steps:

[0082] The experimental data are shown in Table 5 below:

[0083] Table 5, Standard Curve of Comparative Example 2

[0084]

[0085] The relationship between the measured aflatoxin spore concentration and the percentage absorbance value at 450 nm is as Figure 8 shown and in Table 6.

[0086] Table 6, Comparative Example 2 - Spore Concentration and Percentage Absorbance Value

[0087]

[0088] It was measured that this kit can detect aflatoxin when the spore concentration is 10 6 and above, and the detection limit of this kit is 0.05 ng / mL. It can be seen that when the spore concentration is 6.20×10 5 per mL, the detection results are not reliable.

[0089] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A fluorescence quantitative PCR method for detecting the spore concentration of aflatoxin-producing strains of Aspergillus flavus in agricultural product samples, comprising the following steps: 1) Shake and mix the sample to be tested with sterilized distilled water to obtain a spore suspension of aflatoxin-producing strains of Aspergillus flavus; transfer the obtained spore suspension of aflatoxin-producing strains of Aspergillus flavus into a 50 mL centrifuge tube and shake and mix for 3 min; filter the mixed spore suspension in the centrifuge tube with sterilized absorbent cotton to remove other substances, then rinse the absorbent cotton with 5 mL of ddH2O to obtain a pure spore suspension, centrifuge at 12000 rpm for 15 min in a centrifuge, discard the supernatant, and add 1 mL of ddH2O to obtain a concentrated spore suspension; 2) Extract the DNA of the concentrated spore suspension of the sample to be tested; the method for extracting the DNA of the sample to be tested is that the spore suspension of the sample to be tested is placed in a 1.5 mL centrifuge tube, centrifuged at 12000 rpm for 15 min in a centrifuge, and the supernatant is discarded; freeze the spore precipitate in liquid nitrogen, grind it, add 200 μL of ddH2O to the centrifuge tube, place it in boiling water for 9 min, centrifuge at 14000 r, 4 °C for 15 min, and the obtained supernatant is the Aspergillus flavus spore DNA solution; 3) Using the DNA of the concentrated spore suspension of the sample to be tested as a template, perform fluorescence quantitative PCR detection with a fluorescence quantitative PCR primer set to obtain a Ct value; the fluorescence quantitative PCR primer set is composed of the nucleotide shown in Sequence 1 in the sequence listing and the nucleotide shown in Sequence 2 in the sequence listing; 4) Substitute the Ct value obtained in step 3) into the linear curve of the spore concentration of Aspergillus flavus toxin-producing strains and the Ct value obtained in step 1), y = -2.7406x + 48.722, where y is the fluorescence quantitative PCR Ct value, and x = lgA, where A is the spore concentration of the aflatoxin-producing strain of Aspergillus flavus, with the unit of number / ml. The spore concentration of the aflatoxin-producing strain of Aspergillus flavus in the spore suspension of the sample to be tested is obtained through conversion; The reaction system of the fluorescence quantitative PCR is a 20 μL system, including 1 μL of DNA template, 0.4 μL of the primer shown in Sequence 1 in the sequence listing at 10 μM, 0.4 μL of the primer shown in Sequence 2 in the sequence listing at 10 μM, 10 μL of Mix, and 8.2 μL of ddH2O; The reaction program of the fluorescence quantitative PCR is: first at 95 °C for 2 min, then at 95 °C for 10 s, 62.5 °C for 30 s, for 40 cycles, and finally Melt Curve Stage: 95 °C for 15 s, 60 °C for 1 min, 95 °C for 1 s.

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