CN113930542A - Primer probe combination and kit for detecting aflatoxin-producing fungi in food - Google Patents
Primer probe combination and kit for detecting aflatoxin-producing fungi in food Download PDFInfo
- Publication number
- CN113930542A CN113930542A CN202111428045.5A CN202111428045A CN113930542A CN 113930542 A CN113930542 A CN 113930542A CN 202111428045 A CN202111428045 A CN 202111428045A CN 113930542 A CN113930542 A CN 113930542A
- Authority
- CN
- China
- Prior art keywords
- primer
- probe
- seq
- producing fungi
- aflatoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—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
-
- 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]
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention provides a primer probe combination and a kit for detecting aflatoxin-producing fungi in food, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, and the primer combination is characterized in that the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, the sequence of the downstream primer is shown as SEQ ID No.2, the probe is shown as SEQ ID No.3, the 5 'end of the probe sequence is modified with a fluorescent group, and the 3' end of the probe sequence is modified with a substance of a fluorescence quenching group; the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1. The primer probe combination and the kit provided by the invention have the advantages of strong specificity, high sensitivity, rapidness and environmental protection in a testing method, and are suitable for rapidly detecting main toxin-producing fungi in food in a basic laboratory.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a primer probe combination and a kit for detecting aflatoxin-producing fungi in food.
Background
Fungi are widely distributed in all levels of food chains in nature, are various in variety, large in quantity and very closely related to human beings, and under the condition of proper temperature and humidity, some toxin-producing fungi can grow and propagate in food (raw materials and processed products), food and feed to produce mycotoxin. Mycotoxins are commonly present in agricultural and sideline products or animal feeds, and can cause acute or chronic poisoning of human beings and animals after being eaten by mistake, damage liver, kidney, nerve tissue, hematopoietic tissue and skin tissue of organisms, teratogenesis, carcinogenesis and the like.
Aflatoxin (AFT) is a mycotoxin with high toxicity and carcinogenicity, which can easily contaminate various foods and agricultural products, and is mainly a secondary metabolite of aspergillus flavus and aspergillus parasiticus. The probability of food and feed being contaminated by AFT is high, and the food and feed can directly or indirectly enter the human food chain, and when people eat food containing AFT, the health of people can be seriously harmed. The AFT mainly pollutes products such as peanuts, corns, cottonseeds, poultry eggs, wheat, milk, dairy products and the like, the maximum allowable residual quantity standard of the AFT in food and feed is recommended by the world health organization, the maximum allowable residual quantity standard is also set in China, the United states and European Union in succession, and the pollution of the AFT is seriously considered by countries in the world.
The detection method of the toxin-producing fungi at present mainly comprises a traditional microorganism culture method and a common PCR method, wherein the traditional microorganism culture method is based on morphological observation, the toxin-producing fungi are classified and identified according to characteristics such as spore morphology, hypha infection, spore production mode and the like, and the common PCR detection process needs to analyze and verify products after amplification. The two methods have the limitations of complicated steps and long inspection period in operation, and are difficult to apply in emergency monitoring. The real-time fluorescence PCR method is used for detecting toxin-producing fungi, can well make up the defects of the traditional method in detection work, greatly shortens the detection time, simplifies the operation flow and meets the requirement of quickly detecting the toxin-producing fungi of import and export foods.
Therefore, based on the advantages of the real-time fluorescence PCR method and the requirement for rapid detection of aflatoxin-producing fungi, a rapid and convenient detection method for aflatoxin-producing fungi is developed to meet the requirement of rapid detection of main toxin-producing fungi in food in basic laboratories.
Disclosure of Invention
In view of the above, the invention provides a novel primer probe combination and a kit for detecting aflatoxin-producing fungi in food. The rapid detection method for aflatoxin-producing fungi can meet the requirement of primary laboratories on rapid detection of main toxin-producing fungi in food.
The technical scheme of the invention is as follows:
a primer probe combination for detecting aflatoxin-producing fungi in food, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, the sequence of the downstream primer is shown as SEQ ID No.2, the probe is shown as SEQ ID No.3, the 5 'end of the probe sequence is modified with a fluorescent group, and the 3' end is modified with a substance with a fluorescence quenching group; the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
The method specifically comprises the following steps:
upstream primeraflR-F1，SEQ ID No.1：5’- CGCCTCATGCTCATACCC-3’；
Downstream primeraflR-R1，SEQ ID No.2：5’- GAGACGCTACTGCTACCAT-3’；
Probe needleaflR-P1，SEQ ID No.3：5’-FAM- AATCAACCACCACACGCTCT-BHQ1-3’。
The invention also provides a primer probe combination for detecting the aflatoxin-producing fungi in food, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.4, the sequence of the downstream primer is shown as SEQ ID No.5, the probe is shown as SEQ ID No.6, the 5 'end of the probe sequence is modified with a fluorescent group, the 3' end is modified with a substance of a fluorescence quenching group, the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
The method specifically comprises the following steps:
upstream primeromt-1-F2，SEQ ID No.4：5’- CACCGAAGCCACGAAG -3’；
Downstream primeromt-1-R2，SEQ ID No.5：5’- GCTGAGATGACCACGAC -3’；
Probe needleomt-1-P2，SEQ ID No.6：5’-FAM- CCGCCGACATCGACAAC -BHQ1-3’。
The invention also provides a primer probe combination for detecting the aflatoxin-producing fungi in food, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.7, the sequence of the downstream primer is shown as SEQ ID No.8, the probe is shown as SEQ ID No.9, the 5 'end of the probe sequence is modified with a fluorescent group, the 3' end is modified with a substance of a fluorescence quenching group, the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
The method specifically comprises the following steps:
upstream primerver-1-F2，SEQ ID No.7：5’-CCCAAACATGCCGTATACTCCGGGT-3’；
Downstream primerver-1-R2，SEQ ID No.8：5’-CTCCAGGAGCCACCGCATT-3’；
Probe needlever-1-P2，SEQ ID No.9：5’-FAM-CCATGCAGCGAACAAAGGTGTCA-BHQ1-3’。
In the invention, three key genes in the aflatoxin biochemical synthesis process are respectively selected: regulatory geneaflRAnsetromycin methoxymethylaseomt-1And variolothricin A dehydrogenasever-1Whether the strain to be detected can produce the aflatoxin or not is judged. Searching a target gene sequence through an NCBI online tool, performing sequence analysis and comparison by using DNAMAN software, selecting a sequence with species specificity and high conservation, respectively designing a primer and a probe combination by using Oligo7 software, wherein the 5 'end of the probe is marked with FAM, and the 3' end of the probe is marked with BHQ 1.
The invention also provides a kit for detecting aflatoxin-producing fungi in food, which is characterized by comprising the following components: any one of the primer probe combination, the positive control and the PCR reaction system.
Further, the positive control is selected from 5.8S rDNA common to fungiITSA gene.
The primer probe combinations of the positive control are respectively shown as SEQ ID NO. 10-12.
The method specifically comprises the following steps:
upstream primerITS-F1，SEQ ID No.10：5’-CTCTTGGTTCCGGCATC-3’；
Downstream primerITS-R1，SEQ ID No.11：5’-TGCGTTCAAAGACTCGAT-3’；
Probe needleITS-P1，SEQ ID No.12：5’-FAM-AGCGAAATGCGATACGTAATGTGA-BHQ1-3’。
In the present invention, 5.8S rDNA common to fungi is usedITSThe sequence is used as a positive reference to judge whether the extracted DNA template and the fluorescence PCR reaction are proper.
Further, the reaction system comprises 12.5 muL 2x Superstart premix Plus (Probe qPCR), 1 muL of each primer Probe and 5 muL of DNA template, and the total volume is complemented to 25 muL by double distilled water.
Further, the final concentration of the primer probes is 0.4 [ mu ] mol/L.
Further, the kit has the following amplification reaction conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 45s, and collecting fluorescence signals; 45 cycles.
The real-time fluorescent PCR technology is that on the basis of PCR, one oligonucleotide probe matched with template DNA and with fluorescent labels in two ends is added. Every time the PCR is circulated, the number of the synthesized new strands corresponds to the released fluorescent group, namely the amount of the PCR product corresponds to the intensity of the fluorescent signal. When the fluorescence signal exceeds a set threshold value, the fluorescence signal can be detected, and the increase of the fluorescence group detected by the instrument can indirectly reflect the amplification amount of the target fragment. And carrying out real-time fluorescence PCR amplification on the template DNA of the sample, and observing an amplification curve of the real-time PCR, thereby rapidly detecting the toxin-producing fungi in the food.
In the invention, aspergillus has various varieties, and the nucleic acid sequence homology between species and between species (and other genera such as candida, cryptococcus and the like) is higher, and meanwhile, the probability of variation of the whole fungus genome is far greater than that of a human genome, so that the gene sequences which can be selected as target gene sequences for species detection are not many, and therefore, the difficulty in designing primers and probes for specifically detecting the fluorescence PCR of common aspergillus such as aspergillus flavus, aspergillus parasiticus and the like is higher. The inventor finds the optimal primer probe pair through a large number of creative tests. The inventor analyzes more than 20 enzymes which play a role in the biosynthesis process of aflatoxin from acetyl coenzyme A, and finds that ver-1 gene codes variolomycin A dehydrogenase to convert the pseudoansamycin, omt-1 gene codes regulatory ansamycin transmethylase to convert the aurothiomycin precursor O-methylaurothiomycin, and gene aflR is used as a regulatory gene to code specific DNA binding protein to activate the aflatoxin biosynthesis pathway. The genes have species specificity and high conservation, so the genes can be used as probes in analyzing genes participating in toxin synthesis or molecular mechanisms participating in regulation and control, and can also be used as specific molecular markers for producing the aflatoxin strains. However, since the specific segment of the target nucleotide is small, a suitable primer probe is designed in the target nucleotide, and only software is used for analysis and comparison, the found primer probe cannot meet the experimental requirements, and a large number of experiments are performed to screen the optimal primer probe pair through manual adjustment and comparison by creative tests.
The primer probe designed by the invention can effectively amplify corresponding target genes, and has strong fluorescent signal for amplifying the target genes, early appearance time and good amplification effect.
The primer probe combination provided by the invention can realize simultaneous detection of aspergillus flavus and aspergillus parasiticus, and has the advantages of strong detection specificity, high sensitivity and high detection speed. The kit can successfully and rapidly detect the main toxin-producing fungi from food samples, has good exclusivity and inclusion, has no cross reaction with common fungi and common pathogenic bacteria, and has the nucleic acid detection sensitivity of key genes of the toxin-producing fungi of 2.0 multiplied by 10-4ng/muL. The detection result is consistent with the result of the common PCR method. The method has the advantages of strong specificity, high sensitivity, rapidness and environmental protection, and is suitable for rapid detection of main toxin-producing fungi in food in primary laboratories.
Drawings
FIG. 1 shows the results of the specificity verification of the primer probes of examples 1 to 3, in which pairs A to D are selected in the order of pairsITS、aflR、omt-1、ver-1The specific detection result of the primer probe, wherein the figure is the serial number of the strain);
FIG. 2 shows nucleic acids of positive strains diluted in a gradientITSThe sensitivity detection result of the gene, wherein A-C in the figure are a fluorescence PCR amplification map, a standard curve and a common PCR electrophoresis chart in sequence;
FIG. 3 shows nucleic acids of positive strains diluted in a gradientaflRThe sensitivity detection result of the gene, wherein A-C in the figure are a fluorescence PCR amplification map, a standard curve and a common PCR electrophoresis chart in sequence;
FIG. 4 shows nucleic acids of positive strains diluted in a gradientomt-1The sensitivity detection result of the gene, wherein A-C in the figure are a fluorescence PCR amplification map, a standard curve and a common PCR electrophoresis chart in sequence;
FIG. 5 shows nucleic acids of positive strains diluted in a gradientver-1The sensitivity detection result of the gene, wherein A-C in the figure are a fluorescence PCR amplification map, a standard curve and a common PCR electrophoresis chart in sequence;
FIG. 6 is the detection spectrum of aflatoxin of a contaminated sample.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A primer probe combination for detecting aflatoxin-producing fungi in food, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, the sequence of the downstream primer is shown as SEQ ID No.2, the probe is shown as SEQ ID No.3, the 5 'end of the probe sequence is modified with a fluorescent group, and the 3' end is modified with a substance with a fluorescence quenching group; the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
The method specifically comprises the following steps:
upstream primeraflR-F1，SEQ ID No.1：5’- CGCCTCATGCTCATACCC-3’；
Downstream primeraflR-R1，SEQ ID No.2：5’- GAGACGCTACTGCTACCAT-3’；
Probe needleaflR-P1，SEQ ID No.3：5’-FAM- AATCAACCACCACACGCTCT-BHQ1-3’。
Example 2
A primer probe combination for detecting aflatoxin-producing fungi in food comprises aflatoxin and aspergillus parasiticus, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID No.4, the sequence of the downstream primer is shown in SEQ ID No.5, the probe is shown in SEQ ID No.6, a substance with a fluorescent group modified at the 5 'end and a fluorescence quenching group modified at the 3' end of the probe sequence, the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
The method specifically comprises the following steps:
upstream primeromt-1-F2，SEQ ID No.4：5’- CACCGAAGCCACGAAG -3’；
Downstream primeromt-1-R2，SEQ ID No.5：5’- GCTGAGATGACCACGAC -3’；
Probe needleomt-1-P2，SEQ ID No.6：5’-FAM- CCGCCGACATCGACAAC -BHQ1-3’。
Example 3
A primer probe combination for detecting aflatoxin-producing fungi in food comprises aspergillus flavus and aspergillus parasiticus, the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.7, the sequence of the downstream primer is shown as SEQ ID No.8, the probe is shown as SEQ ID No.9, a substance with a fluorescent group modified at the 5 'end and a fluorescence quenching group modified at the 3' end of the probe sequence, the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
The method specifically comprises the following steps:
upstream primerver-1-F2，SEQ ID No.7：5’-CCCAAACATGCCGTATACTCCGGGT-3’；
Downstream primerver-1-R2，SEQ ID No.8：5’-CTCCAGGAGCCACCGCATT-3’；
Probe needlever-1-P2，SEQ ID No.9：5’-FAM-CCATGCAGCGAACAAAGGTGTCA-BHQ1-3’。
Example 4
A kit for detecting aflatoxin-producing fungi in food, which is characterized by comprising: any one of the primer probe combination, the positive control and the PCR reaction system.
Further, the positive control is selected from 5.8S rDNA common to fungiITSA gene.
The primer probe combinations of the positive control are respectively shown as SEQ ID NO. 10-12.
The method specifically comprises the following steps:
upstream primerITS-F1，SEQ ID No.10：5’-CTCTTGGTTCCGGCATC-3’；
Downstream primerITS-R1，SEQ ID No.11：5’-TGCGTTCAAAGACTCGAT-3’；
Probe needleITS-P1，SEQ ID No.12：5’-FAM-AGCGAAATGCGATACGTAATGTGA-BHQ1-3’。
In the present invention, 5.8S rDNA common to fungi is usedITSThe sequence is used as a positive reference to judge whether the extracted DNA template and the fluorescence PCR reaction are proper.
Further, the reaction system comprises 12.5 muL 2x Superstart premix Plus (Probe qPCR), 1 muL of each primer Probe and 5 muL of DNA template, and the total volume is complemented to 25 muL by double distilled water.
Further, the final concentration of the primer probes is 0.4 [ mu ] mol/L.
Further, the kit has the following amplification reaction conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 45s, and collecting fluorescence signals; 45 cycles.
The experimental effect is verified:
1. experimental strains: the experiment uses 52 strains (table 1) in total, wherein the strains comprise standard strains of aspergillus flavus, aspergillus parasiticus and fusarium graminearum, other fungus standard strains and other common food-borne pathogenic bacteria standard strains.
TABLE 1 list of strains for experiments
Note: ATCC: american type culture Collection; CMCC: medical microbial strain preservation management center; CGMCC: china general microbiological culture Collection center; and (3) CICC: china center for the preservation and management of industrial microbial strains; ACCC: china agricultural microbial strain preservation management center; CCTCC (China center for type communications): china center for type culture Collection (Wuhan university).
2. Reagents and instrumentation: 2x Superstart premix Plus (Probe qPCR), TAKARA; the goods number is: RR 390; UNlQ-10 columnar fungus genome DNA extraction kit, bio-engineering gmbh, cat no: b511375; the culture media were purchased from Kyork, Guangdong, Microbiol technologies, Inc. 7500 Fast Real Time PCR System, ABI, USA; nanodrop 2000c nucleic acid protein analyzer, Thermo corporation, usa; masteroyoler pro gradient PCR instrument, eppendorf, Germany; QIAxcel Advanced fully automated nucleic acid analysis System, Qiagen, Germany.
3. Extracting genome DNA and determining by a nucleic acid protein analyzer: under aseptic condition, a ring of spores of the target fungi is picked up by the burned inoculating ring and inoculated into a test tube filled with 5mL of the Sachs' culture solution, the temperature is 25 ℃, the speed is 180 r/min, and the shaking table culture is carried out for 48 h. Mycelia (about 0.3 g) were picked from the Saccharum sinensis culture broth and ground into powder by adding liquid nitrogen. Extracting genome DNA by adopting a fungus genome DNA extraction kit, carrying out the extraction step according to the kit instruction, and finally dissolving the nucleic acid in 50 mu L of TE buffer solution. The concentration and purity of the extracted nucleic acid were measured with a ultramicro spectrophotometer (Nanodrop 2000) to ensure that A260/A280 of the extracted nucleic acid was between 1.7 and 1.9. The concentration of DNA was calculated according to formula (1).
ρ= A260×50………………………………（1）
Note: rho-DNA concentration in micrograms per milliliter (ng// μ L), absorbance at A260-260 nm.
4. And (3) common PCR detection: the detection of the aflatoxin-producing fungi (Aspergillus flavus and Aspergillus parasiticus) was performed with reference to the SN/T2583-. The primer sequences and the lengths of the amplified fragments are shown in Table 2.
TABLE 2 toxin-producing fungi common PCR primer sequences
5. Detection of artificially contaminated samples:
weighing 50g of high-quality long-shaped rice, adding 20ml of sterilized water, uniformly mixing, standing overnight, sterilizing at 121 ℃ for 15min, and taking the mixture as a part of added matrix sample. After Aspergillus flavus (CGMCC 3.0117) and Aspergillus parasiticus (CGMCC 3.0124) strains are activated and cultured in a Saccharomyelia liquid culture medium for 7 days, 5mL of culture solution containing hypha/spores are respectively inoculated into a rice culture medium. Preparing 4 artificial pollution samples, namely taking 25g of each sample to prepare and culture the samples according to the method in GB 4789.15; taking out the cultured suspicious fungi, picking out a ring of spores by using the burned inoculating rings under aseptic conditions, inoculating the spores into a test tube filled with 5mL of Sabouraud's liquid culture medium, and culturing for 48h by shaking at 25 ℃ and 180 r/min. Extracting genome DNA by using a fungal genome DNA extraction kit, and detecting aflatoxin-producing fungi (aspergillus flavus and aspergillus parasiticus) by using a fluorescence PCR method. ② after culturing the rest samples for 7d under the conditions of 28 ℃ and 60 percent of relative humidity, taking 5G of each sample, and measuring the aflatoxin according to GB 5009.22-2016 (measuring of B group and G group of aflatoxin in national standard food for food safety).
6. Detection of natural samples:
and (3) extracting 30 representative food and grain samples, culturing fungi according to GB 4789.15-2016, extracting fungal nucleic acid from suspicious bacteria, detecting toxin-producing fungi by respectively adopting the kit of the embodiment 4 of the invention and the common PCR method, and comparing detection results.
7. And (4) analyzing results:
7.1, primer probe specificity test: primer probe specificity tests include inclusion tests and exclusion tests. For ensuring the specificity of the amplification reaction, selectedITSF1/R1/P1 shows that the DNA of all strains in the strain table 1 is detected, the result shows that the fungus DNA has a remarkable amplification curve, other microorganisms are detected to be negative, and the primer probe set has good specificity (figure 1A).
Selected from those in examples 1 to 3 of the present inventionaflR-F1/R1/P1、omt-1-F2/R2/P2、ver-1The F2/R2/P2 primer probe group detects DNA of 5 strains of aspergillus flavus and aspergillus parasiticus standard strains, common aspergillus fungi and other non-aspergillus fungi listed in the table 1, and as a result, only 5 strains of the fungi producing aflatoxin show positive amplification signals, and the other common aspergillus fungi and the non-aspergillus fungi are negative in detection (figure 1B, C, D). Show thataflR-F1/R1/P1、omt-1-F2/R2/P2、ver-1The F2/R2/P2 primer probe set has good specificity.
Table 3 results of specificity verification for primer probes
7.2, detection-sensitivity verification of gradient dilution fungal nucleic acid: and (3) carrying out gradient dilution detection on the fungal nucleic acid by using the screened primer group and an optimized reaction system. Genomic nucleic acid (20 ng/. mu.L) from a positive standard strain was diluted 10-fold in a gradient to 10-1 - 10-6Corresponding to a nucleic acid concentration of 2.0, 2.0X 10-1、2.0×10-2、2.0×10-3、2.0×10-4、2.0×10-5ng/muL. Aspergillus flavus CGMCC 3.0117 nucleic acid extraction for detectionITS、aflR、omt-1、ver-1A gene. The detection is carried out by using a fluorescence PCR method and a common PCR method simultaneously.
ITSThe gene detection result shows that: the detection sensitivity reaches 10-6Dilution (2.0X 10)-5ng/mu L) of 10-1To 10-6The detection results are in a good linear relationship (R2 = 0.999) within the range, the slope of the standard curve is-3.336, and the amplification efficiency reaches 99.4% (FIG. 2). The sensitivity of the corresponding common PCR method reaches 10-5The dilution and the result show that the fluorescence PCR method has higher sensitivity.
aflR、omt-1、ver-1The gene detection sensitivity was 10-5Dilution (2.0X 10)-4 ng/mu L) of 10-1To 10-5The detection results in a good linear relationship within the range, R2 is more than 0.990, the slope of the standard curve is between 3.2 and 3.5, and the amplification efficiency is more than 95 percent (figure 3-5). The sensitivity of the corresponding common PCR method reaches 10-4The dilution and the result show that the sensitivity of the fluorescence PCR method is 2.0 multiplied by 10-4ng/muL, higher than the sensitivity of the common PCR method.
TABLE 4 Aspergillus flavus genomic DNA assay results on gradient dilution
7.3, detection of artificially contaminated samples: according to the culture result of the artificial pollution sample, extracting the nucleic acid of the suspected fungus and then carrying out fluorescence PCR detection to detect the fungus which is suspected to produce aflatoxinITS、aflR、omt-1、ver-1The genes are detected in a table 5, and key genes (key genes) for producing aflatoxin are detected from suspicious bacteria separated from samples polluted by aspergillus flavus CGMCC 3.0117 and aspergillus parasiticus CGMCC 3.0124aflR、omt-1、ver-1). Consistent with the expected results.
TABLE 5 fluorescent PCR detection of suspected fungi in artificially contaminated samples and detection results of toxin production
The fungal toxin is detected by taking the culture of the contaminated bacteria sample, and the result is shown in figure 6, and the aflatoxin is detected in the samples of the contaminated aspergillus flavus CGMCC 3.0117 and the aspergillus parasiticus CGMCC 3.0124.
7.4, detection of natural samples: extracting 30 representative food and grain samples (table 6), culturing fungi according to GB 4789.15-2016, extracting fungal nucleic acid from suspicious bacteria, detecting toxin-producing fungi by the fluorescent PCR method and the common PCR method, wherein the results are shown in table 6, and the results show that the detection results of the fluorescent PCR method are consistent with the detection results of the common PCR.
TABLE 6 Natural sample test results
Therefore, the primer probe combination and the kit can successfully and rapidly detect the main toxin-producing fungi from the food sample, and the method has good exclusivity and inclusivity and is the same as the methodCommon fungi and common pathogenic bacteria have no cross reaction, and the nucleic acid detection sensitivity of key genes of toxin-producing fungi is 2.0 x 10-4ng/muL. The detection result is consistent with the result of the common PCR method. The method has the advantages of strong specificity, high sensitivity, rapidness and environmental protection, and is suitable for rapidly detecting the main toxin-producing fungi in the food in a basic laboratory.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art. It should be noted that the technical features not described in detail in the present invention can be implemented by any prior art in the field.
Sequence listing
<110> North Arch customs technology center
<120> primer probe combination and kit for detecting aflatoxin-producing fungi in food
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cgcctcatgc tcataccc 18
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gagacgctac tgctaccat 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aatcaaccac cacacgctct 20
<210> 4
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctgagatga ccacgac 17
<210> 6
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccgccgacat cgacaac 17
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cccaaacatg ccgtatactc cgggt 25
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctccaggagc caccgcatt 19
<210> 9
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccatgcagcg aacaaaggtg tca 23
<210> 10
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctcttggttc cggcatc 17
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgcgttcaaa gactcgat 18
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
agcgaaatgc gatacgtaat gtga 24
Claims (9)
1. A primer probe combination for detecting aflatoxin-producing fungi in food, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, and is characterized in that the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, the sequence of the downstream primer is shown as SEQ ID No.2, the probe is shown as SEQ ID No.3, the 5 'end of the probe sequence is modified with a fluorescent group, and the 3' end of the probe sequence is modified with a substance of a fluorescence quenching group; the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
2. A primer probe combination for detecting aflatoxin-producing fungi in food is disclosed, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, and the primer combination is characterized in that the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.4, the sequence of the downstream primer is shown as SEQ ID No.5, the probe is shown as SEQ ID No.6, a substance with a fluorescent group modified at the 5 'end and a fluorescence quenching group modified at the 3' end of the probe sequence, the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
3. A primer probe combination for detecting aflatoxin-producing fungi in food is disclosed, wherein the aflatoxin-producing fungi comprise aspergillus flavus and aspergillus parasiticus, and the primer combination is characterized in that the primer comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.7, the sequence of the downstream primer is shown as SEQ ID No.8, the probe is shown as SEQ ID No.9, a substance with a fluorescent group modified at the 5 'end and a fluorescence quenching group modified at the 3' end of the probe sequence, the fluorescent group is FAM, and the fluorescence quenching group is BHQ 1.
4. A kit for detecting aflatoxin-producing fungi in food, which is characterized by comprising: a primer probe combination according to any one of claims 1 to 3, a positive control and a PCR reaction system.
5. The kit for detecting aflatoxin-producing fungi in food products of claim 4 wherein the positive control selects 5.8S rDNA common to fungiITSA gene.
6. The kit for detecting aflatoxin-producing fungi in foods according to claim 5, wherein the primer probe combinations of the positive control are respectively shown in SEQ ID No. 10-12.
7. The kit for detecting aflatoxin-producing fungi in food according to claim 4, wherein the reaction system comprises 12.5 μ L2 x Superstart premix Plus 8, 1 μ L of each primer probe, 5 μ L of DNA template, and the total volume is made up to 25 μ L by double distilled water.
8. The kit for detecting aflatoxin-producing fungi in foods according to claim 7, wherein the final concentration of the primer probes is 0.4 μmol/L.
9. The kit for detecting aflatoxin-producing fungi in foods according to claim 8, wherein the amplification reaction conditions of the kit are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 45s, and collecting fluorescence signals; 45 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111428045.5A CN113930542A (en) | 2021-11-29 | 2021-11-29 | Primer probe combination and kit for detecting aflatoxin-producing fungi in food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111428045.5A CN113930542A (en) | 2021-11-29 | 2021-11-29 | Primer probe combination and kit for detecting aflatoxin-producing fungi in food |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113930542A true CN113930542A (en) | 2022-01-14 |
Family
ID=79288496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111428045.5A Pending CN113930542A (en) | 2021-11-29 | 2021-11-29 | Primer probe combination and kit for detecting aflatoxin-producing fungi in food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113930542A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6623932B1 (en) * | 2002-03-26 | 2003-09-23 | Council Of Scientific And Industrial Research | Primers for identifying aflatoxinogenic aspergilli and an improved method thereof |
| CA2480472A1 (en) * | 2002-03-26 | 2003-10-02 | Council Of Scientific And Industrial Research | Novel primers for identifying aflatoxinogenic aspergilli and an improved use thereof |
| CN106318938A (en) * | 2015-06-26 | 2017-01-11 | 中粮集团有限公司 | Single PCR detection primer pair group for fungus producing aflatoxin, detection method and application thereof |
| CN106318939A (en) * | 2015-06-26 | 2017-01-11 | 中粮集团有限公司 | Multiple PCR detection primer group, kit and detection method for fungus producing aflatoxin |
| CN108070675A (en) * | 2018-02-10 | 2018-05-25 | 杭州缔蓝生物技术有限公司 | Primer combination of probe and PCR kit for fluorescence quantitative a kind of while that detect three kinds of aspergillus |
-
2021
- 2021-11-29 CN CN202111428045.5A patent/CN113930542A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6623932B1 (en) * | 2002-03-26 | 2003-09-23 | Council Of Scientific And Industrial Research | Primers for identifying aflatoxinogenic aspergilli and an improved method thereof |
| CA2480472A1 (en) * | 2002-03-26 | 2003-10-02 | Council Of Scientific And Industrial Research | Novel primers for identifying aflatoxinogenic aspergilli and an improved use thereof |
| CN106318938A (en) * | 2015-06-26 | 2017-01-11 | 中粮集团有限公司 | Single PCR detection primer pair group for fungus producing aflatoxin, detection method and application thereof |
| CN106318939A (en) * | 2015-06-26 | 2017-01-11 | 中粮集团有限公司 | Multiple PCR detection primer group, kit and detection method for fungus producing aflatoxin |
| CN108070675A (en) * | 2018-02-10 | 2018-05-25 | 杭州缔蓝生物技术有限公司 | Primer combination of probe and PCR kit for fluorescence quantitative a kind of while that detect three kinds of aspergillus |
Non-Patent Citations (1)
| Title |
|---|
| 秦文彦;程洁;应盛华;冯明光;: "可污染食品及饲料的产黄曲霉毒素真菌的多重PCR检测" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Desjardins et al. | Wheat kernel black point and fumonisin contamination by Fusarium proliferatum | |
| CN109593868B (en) | Characteristic nucleotide sequence for detecting pseudomonas bacteria, and specific primer, kit and detection method thereof | |
| Sukmawati et al. | Isolation and characterization of aflatoxigenic Aspergillus spp. from maize of livestock feed from Bogor | |
| CN111154900A (en) | Pseudomonas aeruginosa specific new molecular target and rapid detection method thereof | |
| Ramachandran et al. | Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis | |
| CN112226523B (en) | Specific detection probe and kit for acetic acid-resistant lactobacillus and application of specific detection probe and kit | |
| CN113930543A (en) | Real-time fluorescent PCR (polymerase chain reaction) kit for detecting fusarium graminearum toxigenic fungi in food | |
| CN110951898B (en) | New specific molecular target of 4 species in Cronobacter and rapid detection method thereof | |
| CN112646904A (en) | Detection method of Burkholderia gladioli and acid-produced strain of Miermentaria, fluorescent PCR primer and probe for detection | |
| Pavlović et al. | Morphology, pathogenicity and molecular identification of Fusarium spp. associated with anise seeds in Serbia. | |
| CN110846423A (en) | Fluorescent quantitative PCR (polymerase chain reaction) rapid detection method for pseudomonas fluorescens, kit and application | |
| CN113957164B (en) | CRISPR One post detection method and kit thereof for Cronobacter in infant formula powder | |
| CN113930542A (en) | Primer probe combination and kit for detecting aflatoxin-producing fungi in food | |
| CN115198026A (en) | Burkholderia gladioli and coconut toxin pathogenic variety specific new molecular target and rapid detection method thereof | |
| CN112176080B (en) | Nested PCR primer group, kit and detection method for specifically detecting purple sisal leaf roll disease phytoplasma | |
| CN103361418A (en) | Preparation method and application of nucleic acid fingerprint characteristic spectrum based on bacteria of 16S rDNA | |
| JP2006238838A (en) | Method for assaying existence of bacterium | |
| CN103060431A (en) | 16S rDNA based preparation method of bacteria nucleic acid fingerprint characteristic spectrums and application thereof | |
| CN103352257A (en) | Method for preparing bacterial nucleic acid fingerprint characteristic spectrum library | |
| JP4117475B2 (en) | Primer set and bacteria detection method | |
| CN111518927A (en) | TaqMan probe quantitative detection method for detecting pseudomonas putida and corresponding kit | |
| CN114107532B (en) | Molecular target for identifying pseudomonas aeruginosa and quantitative detection method thereof | |
| CN114107526B (en) | RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof | |
| CN103060926A (en) | Enzyme digestion based preparation method of bacteria nucleic acid fingerprint characteristic spectrums and application thereof | |
| CN114277166B (en) | RPA detection primer, probe and detection method for melon bacterial fruit blotch |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |