CN114686617B - Method for detecting fumonisin synthetic gene in aspergillus niger group strain - Google Patents
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Abstract
The invention provides a detection method of fumonisin synthetic genes in an Aspergillus niger group strain. The detection method comprises the following steps: detecting by adopting a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in a sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table, the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, and the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table. The detection method can detect when the Aspergillus niger group strains do not produce FB and the FB production level is extremely low by using a PCR technology, and can realize early warning of the FB production pollution risk of the Aspergillus niger group strains.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection method of fumonisin synthetic genes in aspergillus niger group strains.
Background
Fumonisins (B type fumonisins, FB) are structurally similar diester compounds composed of different polyhydric alcohols and tricarballylic acid, and FB has FA 1 、FA 2 、FB 1 、FB 2 、FB 3 、FB 4 、FC 1 、FC 2 、FC 3 、FC 4 And FP 1 The total number is 11. FB has hepatorenal toxicity, can cause equine brain softening, and is classified as a class 2B carcinogen by the International Agency for Research on Cancer (IARC). The aspergillus niger group strain is a common strain in the food fermentation industry, but after FB is detected in aspergillus niger fermentation liquor for the first time, the safety problem of the aspergillus niger group strain is widely concerned again.
An important current method for determining whether a strain produces FB is by determining whether FB can be produced after fermentation by a strain of the Aspergillus niger group. The commonly adopted technology mainly comprises a chromatographic technology, a chromatographic-mass spectrometry combined technology and the like. Although the chromatographic technique and the chromatographic-mass spectrometry combined technique can carry out qualitative or quantitative analysis on the pollution condition of FB in food or feed, when the two methods are used for detection, the detection can be carried out only when FB is generated or polluted to a certain degree in a sample, but the detection cannot be carried out when the FB is not generated or the FB level is extremely low in the Aspergillus niger strain, and the prediction of the FB pollution risk of the Aspergillus niger strain and the early warning cannot be realized.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting a fumonisin synthetic gene in an Aspergillus niger group strain.
The embodiment of the invention provides a detection method of fumonisin synthetic genes in an Aspergillus niger group strain, which comprises the following steps:
and (2) detecting by using a first forward primer and a first reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in the sequence table, and the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table.
Specifically, the detection method further comprises: a second forward primer and a second reverse primer, wherein the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, and the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table.
Specifically, the detection method comprises the following steps:
extracting total DNA of a sample to be detected, and purifying the total DNA;
after the purity of the purified total DNA is determined, performing amplification by using a polymerase chain reaction by using the first forward primer, the first reverse primer, the second forward primer and the second reverse primer to obtain an amplification product;
carrying out agarose gel electrophoresis on the amplification product and the nucleic acid molecular weight reference substance to obtain an amplification fragment;
if the amplified fragment is at 749bp, the aspergillus niger group bacterial strain fumonisin synthetic gene exists in the sample to be detected.
Specifically, the system for amplification comprises: 1-2 muL of the total DNA, 1.0 muL of the first forward primer, 1.0 muL of the first reverse primer, 1.0 muL of the second forward primer, 1.0 muL of the second reverse primer, 12.5 muL of a 2 XHi-Fi DNA polymerase mixture and ddH 2 O 8.5～9.5μL。
Specifically, the procedure of amplification comprises: pre-denaturation at 95 ℃ for 2min, 30 cycles, extension at 72 ℃ for 5min, each of said cycles comprising: denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s and extension at 72 ℃ for 60 s.
Specifically, the total DNA is extracted from the sample to be tested after culturing.
Further, the culture method comprises: inoculating the sample to be detected on a culture medium, and performing static culture in a constant-temperature incubator at the temperature of 28 +/-1 ℃ for 5-7 days to obtain a culture;
and (3) selecting the culture, inoculating the culture in a liquid culture medium, and performing shake culture at 28 +/-1 ℃ for 24-120 h.
Specifically, the detection method further comprises: the purity of the purified total DNA was determined using a spectrophotometer.
The embodiment of the invention provides a method for detecting a fumonisin synthetic gene in an Aspergillus niger group strain,sdr1the gene is a key gene for FB synthesis, andsdr1the gene is a strain of the Aspergillus niger groupfumGenes specific to the cluster, the detection method being directed tosdr1Designing a first forward primer and a first reverse primer according to genes, judging whether a fumonisin synthetic gene in an Aspergillus niger group strain exists in a sample to be detected or not through the first forward primer and the first reverse primer, if the fumonisin synthetic gene in the Aspergillus niger group strain exists, then FB pollution risk exists in the sample to be detected, and if the fumonisin synthetic gene in the Aspergillus niger group strain does not exist, then FB pollution risk does not exist in the sample to be detectedEarly warning is carried out. Since the species of the Aspergillus niger group also includes Aspergillus carbonarius (A. carbonariusAspergillus carbonarius) Aspergillus wenteri (A), (B)Aspergillus wentii Wehmer) Aspergillus brasiliensis (A. brasiliensis: (A. brasiliensis))Aspergillus brasiliensis) A plurality of strains all carryingsdr1Genes and risks producing FB, so the specific first forward primer and the first reverse primer designed by the invention can be used for carrying a plurality of strains in the Aspergillus niger groupsdr1Detecting the strain of the gene, and is not limited to aspergillus niger; at the same time, aim atsdr1The first forward primer and the first reverse primer designed by the gene can effectively avoid the influence of fusarium moniliforme which can also produce FB on detection.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the present invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and are not intended to limit the invention.
Fig. 1 is a diagram of PCR amplification results of a positive control strain, a negative control strain and 4 samples to be tested provided in the embodiment of the present invention, wherein a lane M: marker, lane 1: test sample CFSA03, lane 2: test sample CFSA04, lane 3: test sample CFSA05, lane 4: test sample CFSA06, lane 5: positive control strain, lane 6: negative control strain, lane 7: blank control, lane 8: an ITS fragment.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.
Examples
The embodiment of the invention provides a detection method of fumonisin synthetic genes in an Aspergillus niger group strain, which comprises the following steps:
culturing a sample to be detected to obtain a culture, extracting the total DNA of the sample (culture) to be detected, and purifying the total DNA; in this example, 2 aspergillus niger samples CSFA03 and CFSA04 capable of producing FB and 2 aspergillus niger samples CFSA05 and CFSA06 not producing FB were respectively used as samples to be tested for detection, and the sources of the 4 aspergillus niger samples were all preserved in the microbial chamber of the national food safety risk assessment center. Meanwhile, a positive control strain, a negative control strain and a blank control are used as controls, in the embodiment, the positive control strain is aspergillus niger CFSA01, the negative control strain is aspergillus flavus CFSA02, the blank control is distilled water, and the positive control strain and the negative control strain are stored in a national food safety risk assessment center microorganism room.
The detection is carried out by adopting a first forward primer, a first reverse primer, a second forward primer and a second reverse primer, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in a sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table, the sequence of the second forward primer is shown as SEQ ID NO. 3 in the sequence table, and the sequence of the second reverse primer is shown as SEQ ID NO. 4 in the sequence table.
In this example, the ITS gene sequence of the A.niger group strain was amplified using the ITS gene as an internal reference gene and the second forward primer and the second reverse primer as universal primers.
After the purity of the purified total DNA is determined, a polymerase chain reaction is adopted to amplify by utilizing a first forward primer, a first reverse primer, a second forward primer and a second reverse primer to obtain an amplification product;
carrying out agarose gel electrophoresis on the amplification product and a nucleic acid molecular weight reference substance (DL 2000) together to obtain an amplification fragment;
if the amplified fragment is at 749bp, the fumonisin synthetic gene in the aspergillus niger group bacterial strain exists in the sample to be detected.
If the amplified fragment is not at 749bp, the fumonisin synthetic gene in the Aspergillus niger group strain does not exist in the sample to be detected.
Specifically, the system for amplification comprises: 1-2 muL of total DNA, 1.0 muL of first forward primer, 1.0 muL of first reverse primer, 1.0 muL of second forward primer, 1.0 muL of second reverse primer, 12.5 muL of 2 XHi-Fi DNA polymerase mixture and ddH 2 8.5-9.5 mu L of O, and the total volume of the amplified system is 25 mu L. Wherein the 2 XHi-Fi DNA polymerase mixture comprises: high fidelity DNA polymerase, Mg 2+ (20. mu.M), dNTPs and PCR buffer. In the implementation, the plant genome DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd.) can be used for amplification.
Specifically, the procedure for amplification is shown in table 1.
Table 1 shows the procedure of amplification
Further, the culture method comprises: respectively inoculating a positive control strain, a negative control strain and 5 to-be-detected samples on a sand-protecting agar slant culture medium or a potato glucose agar slant culture medium under an aseptic condition, and performing static culture in a constant-temperature incubator at (28 +/-1) DEG C for 5-7 days to obtain a culture; the culture is selected and inoculated in 5mL of improved LB liquid culture medium, and shake culture is carried out at the temperature of 28 +/-1 ℃ and the rpm of 200/min for 24-120 h.
Specifically, the detection method further comprises: the purity of the purified total DNA was determined using a spectrophotometer. In this example, the purity of total DNA was measured using NanoDrop ND-1000, and A of total DNA of the sample to be tested 260 /A 280 The ratio is in the range of 1.7-1.9.
Specifically, in the electrophoresis, the electrophoresis was carried out for 30min on a 1% agarose gel at a voltage of 120V.
Specifically, the method for extracting the total DNA of the sample to be detected comprises the following steps: placing the cultures in centrifuge tubes respectively, collecting precipitates after high-speed centrifugation, wherein the precipitates are cultured mycelia, and placing the precipitates in a mortar. Liquid nitrogen will be added to the mortar and the precipitate will be ground to a powder, and then the total DNA will be extracted and purified according to the procedure of a commercial plant genome extraction kit (Qiagen).
The result of the detection
FIG. 1 is a diagram showing electrophoresis results of a positive control strain, a negative control strain, a blank control strain and 4 samples to be tested, and it can be seen from FIG. 1 that 2 strains of the group of FB-producing Aspergillus niger amplify target fragments (the amplified fragments are 749 bp), 2 strains of the group of FB-not-producing Aspergillus niger do not amplify target fragments, and all strains are amplified to ITS fragments by a second forward primer and a second reverse primer, which proves that the amplification results of this embodiment are reliable.
The embodiment of the invention provides a method for detecting a fumonisin synthetic gene in an Aspergillus niger group strain,sdr1the gene is a key gene for FB synthesis, andsdr1the gene is a strain of the Aspergillus niger groupfumGenes specific to the cluster, the detection method being directed tosdr1The method comprises the steps of designing a first forward primer and a first reverse primer through genes, judging whether a fumonisin synthetic gene in an Aspergillus niger group strain exists in a sample to be detected or not through the first forward primer and the first reverse primer, wherein if the fumonisin synthetic gene in the Aspergillus niger group strain exists, the sample to be detected has FB pollution risk, and if the fumonisin synthetic gene in the Aspergillus niger group strain does not exist, the sample to be detected does not have FB pollution risk. Since the species of the Aspergillus niger group also includes Aspergillus carbonarius (A. carbonariusAspergillus carbonarius) Aspergillus wenter (A. wenter)Aspergillus wentii Wehmer) Aspergillus brasiliensis (A. brasiliensis: (A. brasiliensis))Aspergillus brasiliensis) The strains which produce the poison carrysdr1Genes and risks of FB production, the specific first forward primer designed by the inventionAnd the first reverse primer can be used for carrying a plurality of strains in the Aspergillus niger groupsdr1Detecting the strain of the gene, and is not limited to aspergillus niger; at the same time, aim atsdr1The first forward primer and the first reverse primer designed by the gene can effectively avoid the influence of fusarium moniliforme which can also produce FB on detection. In addition, the detection method has the advantages of short detection period, high sensitivity, strong specificity, quick and simple operation, and no need of long culture time and expensive instruments and equipment.
Although the embodiments of the present invention have been described above, the above description is only for the convenience of understanding the present invention, and is not intended to limit the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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Claims (7)
1. A detection method of fumonisin synthetic genes in strains of an Aspergillus niger group is characterized by comprising the following steps:
and (2) detecting by adopting a first forward primer and a first reverse primer to obtain an amplification product, wherein the sequence of the first forward primer is shown as SEQ ID NO. 1 in the sequence table, the sequence of the first reverse primer is shown as SEQ ID NO. 2 in the sequence table, and if the length of the amplification product is 749bp, the fumonisin synthetic gene in the Aspergillus niger group strain exists in the sample to be detected.
2. The detection method according to claim 1, characterized in that it comprises:
extracting total DNA of a sample to be detected, and purifying the total DNA;
after the purity of the purified total DNA is determined, performing amplification by using a polymerase chain reaction by using the first forward primer and the first reverse primer to obtain an amplification product;
subjecting the amplification product and a nucleic acid molecular weight reference to agarose gel electrophoresis;
if the amplification product is at 749bp, the to-be-detected sample contains the fumonisin synthetic gene in the aspergillus niger group bacterial strain.
3. The detection method according to claim 2, wherein the amplification system comprises: the total DNA 1-2 mu L, the first forward primer 1.0 mu L, the first reverse primer 1.0 mu L, a 2 XHi-Fi DNA polymerase mixture 12.5 mu L and ddH 2 O 8.5～9.5μL。
4. The detection method according to claim 2, wherein the amplification procedure comprises: pre-denaturation at 95 ℃ for 2min, 30 cycles, extension at 72 ℃ for 5min, each of said cycles comprising: denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s and extension at 72 ℃ for 60 s.
5. The method according to claim 2, wherein the total DNA is extracted from the sample after culturing.
6. The detection method according to claim 5, wherein the culturing method comprises: inoculating the sample to be detected on a culture medium, and performing static culture in a constant-temperature incubator at 28 +/-1 ℃ for 5-7 days to obtain a culture;
and (3) selecting the culture, inoculating the culture in a liquid culture medium, and performing shake culture at 28 +/-1 ℃ for 24-120 h.
7. The detection method according to claim 2, further comprising: the purity of the purified total DNA was determined using a spectrophotometer.
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| CN202210541517.6A CN114686617B (en) | 2022-05-19 | 2022-05-19 | Method for detecting fumonisin synthetic gene in aspergillus niger group strain |
| PCT/CN2022/131072 WO2023221420A1 (en) | 2022-05-19 | 2022-11-10 | Detection method for fumonisin synthetic gene in aspergillus niger colony strain |
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| WO2000046397A1 (en) * | 1999-02-02 | 2000-08-10 | Technion Research And Development Foundation Ltd. | Nucleic acid-based assay and kit for the detection of alternaria contamination in food products |
| FR2834997B1 (en) * | 2002-01-21 | 2004-03-19 | Evialis | METHOD FOR DETECTION OF OCHRATOXIN A OR CITRININ PRODUCING FUNGI |
| US9290817B2 (en) * | 2010-08-19 | 2016-03-22 | Council Of Scientific & Industrial Research | Diagnostic assays for the detection and identification of aspergilli |
| US8956821B2 (en) * | 2013-02-06 | 2015-02-17 | Medical Service Consultation International, Llc | Methods and compositions for detecting Aspergillus terreus, Aspergillus niger, and mycotoxins |
| CN106893762A (en) * | 2015-12-17 | 2017-06-27 | 中国农业科学院植物保护研究所 | A kind of LAMP detection primer combination of fumonisin and its LAMP detection kit and LAMP method |
| CN105886643A (en) * | 2016-05-26 | 2016-08-24 | 长江大学 | PCR (polymerase chain reaction) detection primers and method for Aspergillus fumigatus |
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| CN114686617B (en) * | 2022-05-19 | 2022-08-26 | 国家食品安全风险评估中心 | Method for detecting fumonisin synthetic gene in aspergillus niger group strain |
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