Patent Application
20210189512
The disclosure belongs to the field of biology, and specifically relates to a method for identifying and evaluating the toxigenic capability of aflatoxigenic strain.
Aflatoxin is the most toxic mycotoxin found so far. Taking aflatoxin B1 as an example, its toxigenic capability is 10 times greater than that of potassium cyanide and 68 times greater than that of arsenic. Aflatoxin is classified as a Class I carcinogen by the International Cancer Organization. Aflatoxin can easily contaminate grain and oil products such as peanuts, corn, rice, etc., as well as many plant products such as walnuts, pistachios and Chinese medicinal materials. There have been many human and livestock poisoning incidents caused by aflatoxin in China and other countries. According to the latest report from the International Agency for Research on Cancer (IARC), about 500 million people in developing countries alone are at risk of exposure to aflatoxin. China is an area where there is a high-level of aflatoxin pollution. The results of the census conducted by the Ministry of Agriculture for many years show that the contamination of main agricultural products in China by aflatoxin is gradually increasing, and the content of toxins in highly contaminated areas exceeds the limit by hundreds of times. Although the highly-toxic mycotoxigenic strains account for less than 20%, they have become a major hidden danger that threatens the quality and safety of crop products.
Aflatoxin is mainly produced by fungi such as Aspergillus flavus and Aspergillus parasiticus. Studies have shown that the capabilities of different Aspergillus flavus strains in producing aflatoxin, that is, their toxigenic capability may be hundreds of times different. Strains with toxigenic capability are a major source of high contamination. However, so far there is a lack of effective method for efficiently identifying strains with toxigenic capability by using specificity. At present, there are mainly two methods for identifying the toxigenic capability of Aspergillus flavus strains: one is to evaluate the toxigenic capability of the strains by measuring the aflatoxin yield produced by the strains after a certain period of time of culturing the strain. For one thing, this type of method takes a long time because the strains must be isolated and then cultured, and then the evaluation is conducted by measuring the content of aflatoxin. For another, the biosynthesis of aflatoxin is affected by many and complex factors. Besides, the aflatoxin yield varies greatly from one batch to another batch of the same strains, and the toxigenic capability result is not suitable to be used to characterize the strain's inherent yield characteristics, which affects the accuracy and reliability of the identification and evaluation results of this method. Another type of method is to evaluate the toxigenic capability of strains by measuring the transcriptional quantity of genes related to toxigenic capability. There is literature regarding detection of Nor-1 gene for evaluating toxigenic capability. However, the disadvantage of this type of method is that in the natural state, there are defects in toxigenic genes other than the Nor-1 gene, which leads to the fact that even if the expression of the Nor-1 gene is detected, it does not produce aflatoxin by nature, and thus leading to false results of such methods.
Briefly, objectively and accurately identifying and evaluating the toxigenic capability of aflatoxigenic strain has been an unresolved problem to worldwide practitioners.
In view of the deficiencies of the related art, the disclosure aims to provide a method for identifying and evaluating the toxigenic capability of aflatoxigenic strain.
In order to achieve the purpose of the disclosure, the technical solution adopted by the disclosure is:
A method for identifying and evaluating the toxigenic capability of aflatoxigenic strain includes the following steps, measuring the aflatoxin yield and Nor-1 gene transcriptional quantity to obtain the ratio of the aflatoxin yield to Nor-1 gene transcriptional quantity, identifying and evaluating the toxigenic capability of aflatoxigenic strain according to the ratio of the aflatoxin yield to Nor-1 gene transcriptional quantity.
According to the above solution, the method for measuring the aflatoxin yield includes the following steps: culturing the Aspergillus flavus strain, taking the spores of Aspergillus flavus for shaking culture, after the culture, filtering the filtrate, and measuring the concentration of aflatoxin in the filtrate.
According to the above solution, the medium used for culturing the Aspergillus flavus strain is CDA medium, and the culture conditions are: culture at 28° C. and 90% humidity for 10 days.
The medium used for shaking culture of the spores of Aspergillus flavus is a potato dextrose liquid medium, and the culture conditions are: shaking culture at 28° C. and 200 rpm for 96 hours.
According to the above solution, the immunoaffinity purification-high performance liquid chromatography standard method is adopted to measure the concentration of aflatoxin in the filtrate after shaking culture of the spores of Aspergillus flavus.
According to the above solution, the method for measuring the transcriptional quantity of the Nor-1 gene includes the following steps: culturing the Aspergillus flavus strain, taking the spores of Aspergillus flavus for shaking culture, after completion of the culture, filtering the hyphae of Aspergillus flavus, and obtaining the dried bacteria after drying, and then using the conventional Nor-1 gene transcriptional quantity measuring method to measure the Nor-1 gene transcriptional quantity in the dried bacteria.
According to the above solution, the aflatoxin yield and Nor-1 gene transcriptional quantity can also be obtained by the synchronization detection RT-PCR method. The specific steps include:
(1) Establishment of an S-type standard curve for quantifying aflatoxin: in the immune reaction stage, when the amount of aflatoxin monoclonal antibody 1C11 coating is constant, use aflatoxin standard products of different concentrations to compete with phage V2-5 to be bound to 1C11. After the immune reaction is over, the phage bound to 1C 11 is eluted. Aflatoxin standard products of different concentrations correspond to different amounts of eluted phage. The phage in the eluate will release the DNA molecules during the PCR heating process, and the released DNA molecules are used as the amplification targets in the RT-PCR reaction. Each of the eluate is subjected to the RT-PCR amplification reaction using the kit, and different Ct values are obtained after the amplification reaction is completed. The logarithm of the concentration of aflatoxin is the abscissa, and the Ct value is the ordinate, and regression analysis is performed to obtain the S-type standard curve for quantifying aflatoxin.
(2) Establishment of a RT-PCR standard curve for Nor-1 gene transcriptional quantity: serially dilute samples of known copy number of nor-1 gene DNA fragment Tq-nor1 into different copy numbers, and then use the kit to perform RT-PCR amplification reaction to each copy number Tq-nor1 separately. Different Ct values are obtained after the amplification reaction. The logarithm of the copy number of Tq-nor1 is used as the abscissa and the Ct value is used as the ordinate to perform regression analysis, thereby obtaining standard curve for quantifying nor-1 gene transcription.
(3) Culturing Aspergillus flavus strain, taking the Aspergillus flavus spores for shaking culture. After the culture is completed, filter the strain culture solution and the fungus ball of Aspergillus flavus. After diluting the strain culture solution by a certain ratio, use it to replace the aflatoxin standard products in the immune reaction carried out in step (1) for the immune competition reaction. After the competitive reaction, the phage bound to 1C11 is eluted, and the V2-5 phage in the eluate is used as the amplification template for quantifying aflatoxin in the synchronous RT-PCR amplification reaction. Additionally, after drying the fungus ball of Aspergillus flavus, the total RNA is extracted and reverse transcribed into cDNA, and the cDNA is diluted by a certain ratio and used as a template for the amplification of Nor-1 gene in the synchronous RT-PCR amplification reaction.
(4) Use the V2-5 bacteriophage and the cDNA as templates to perform the synchronous RT-PCR amplification reaction. After the amplification reaction, two Ct values are obtained, and the two Ct values are respectively substituted into the S-type standard curve for quantifying aflatoxin and standard curve for quantifying nor-1 gene transcription, thereby making conversion to obtain the concentration of aflatoxin and nor-1 gene transcriptional quantity, so as to determine the ratio of aflatoxin yield to Nor-1 gene transcriptional quantity.
According to the above solution, the phage surface-displaying aflatoxin anti-idiotypic nano antibody is phage VHH 2-5, and the aflatoxin monoclonal antibody is the aflatoxin monoclonal antibody 1C11.
According to the above solution, in the reaction system of the synchronous RT-PCR amplification reaction, the final concentration of the upstream and downstream primers: Ph-F, Ph-R, Tq-nor1-F, and Tq-nor1-R is 300 to 400 nM; fluorescent probe: the final concentration of Ph-probe and Tq-probe is 200 to 400 nM; final dosage of DNA polymerase: 0.5 U to 1.0 U; final concentration of MgCl2: 1 mM to 2 mM; final concentration of dNTPs: 200 uM to 400 uM.
According to the above solution, the nucleotide sequence of the upstream primer Ph-F is shown in SEQ ID NO. 1; the nucleotide sequence of the downstream primer Ph-R is shown in SEQ ID NO. 2; the nucleotide sequence of fluorescent probe Ph-probe is shown in SEQ ID NO. 3; the nucleotide sequence of the upstream primer Tq-nor1-F is shown in SEQ ID NO. 4; the nucleotide sequence of the downstream primer Tq-nor1-R is shown in SEQ ID NO. 5; the nucleotide sequence of fluorescent probe Tq-probe is shown in SEQ ID NO. 6.
According to the above solution, the reaction system of the synchronous RT-PCR amplification reaction includes: universal probe qPCR master mix 5 μL, Ph-F 0.1 μL, Ph-R 0.1 μL, Ph-probe 0.1 μL, phage template 2 μL, Tq-nor1-F 0.1 μL, Tq-nor1-R 0.1 μL, Tq-probe 0.1 μL, Nor-1 gene template 1 μL, DNA polymerase 0.2 μL, MgCl2 0.84, dNTPs 0.2 μL, add H2O to make it up to 10 μL.
According to the above solution, the conditions of the RT-PCR synchronous amplification reaction are 95° C., 5 min; 95° C., 10 s, 60° C., 30 s, 40 cycles.
According to the above solution, the concentration range of aflatoxin in the S-type standard curve for quantifying aflatoxin is 33.33 ng/mL to 1.69 pg/mL, and the lowest detection line LOD of aflatoxin is 0.018 ng/mL. In the standard curve for quantifying nor-1 gene transcription, the copy number of nor-1 gene ranges from 102 to 108.
According to the above solution, the toxigenic capability of the aflatoxigenic strain is defined as Y, and the ratio of aflatoxin yield to Nor-1 gene transcriptional quantity (AFT/Nor-1) is defined as X. The formula for identifying toxigenic capability of Aspergillus flavus is Y=10.14X−16.20. For highly toxigenic strain, the toxigenic capability is >150 ng/mL; the medium toxigenic capability: 50<the toxigenic capability<150 ng/mL; the low toxigenic capability: the toxigenic capability<50 ng/mL; no toxin: 0. Substitute the above values into the formula to calculate the identification range of toxigenic capability: AFT/Nor-1>16.4, which indicates a highly toxigenic strain; 6.5<AFT/Nor-1<16.4, which indicates a medium toxigenic strain; 0<AFT/Nor-1<6.5, which indicates a low toxigenic strain; AFT/Nor-1=0, which indicates a non-toxigenic strain.
The advantageous effect of the disclosure is as follows.
(1) The research of the disclosure has proved that the ratio of the aflatoxin yield to the Nor-1 gene transcriptional quantity is of high relative stability. By establishing the identification model of the toxigenic capability of Aspergillus flavus strains, the regression equation for the ratio of toxigenic capability of Aspergillus flavus and aflatoxin yield to nor-1 gene transcriptional quantity (AFT/Nor-1) can be obtained. By determining the AFT/Nor-1 ratio, it is possible to realize fast and accurate identification and evaluation of toxigenic capability of Aspergillus flavus strains, and which is of great significance to the early warning and prevention and controlling of aflatoxin pollution.
(2) The disclosure establishes a synchronous RT-PCR detection method for synchronously detecting the toxigenic capability of Aspergillus flavus strains and the nor-1 gene transcriptional quantity. There is a good linear relationship between the toxigenic capability of Aspergillus flavus strains and nor-1 gene transcriptional quantity obtained by using the synchronous detection RT-PCR method of the disclosure and the aflatoxin quantified through HPLC as well as the Nor-1 gene transcriptional quantity quantified through Nanodrop. Therefore, the result obtained for the ratio of AFT/Nor-1 (toxigenic capability of Aspergillus flavus strain/nor-1 gene transcriptional quantity) obtained based on the synchronous detection RT-PCR method is reliable and accurate, and can be used as an identification index to determine the toxigenic capability of Aspergillus flavus strain.
(3) In the synchronous detection RT-PCR method for toxigenic capability of Aspergillus flavus strain and nor-1 gene transcriptional quantity described in the disclosure, the required amount of reagents is less, and the cost is lower, and therefore high-throughput detection can be achieved. The synchronous detection RT-PCR method simplifies the analysis mode, optimizes the experimental process and structure, and provides a detection platform and theoretical basis for the synchronous analysis of aflatoxin and other small molecules in its synthesis pathway.
In order to make the disclosure more comprehensible, the content of the disclosure will be further clarified below in conjunction with the embodiments, but the content of the disclosure is not limited to the following embodiments.
1. The Study Proved that the Ratio of Aflatoxin Yield to Nor-1 Gene Transcriptional Quantity is of High Relative Stability.
Weigh 1 g of NaNO3, 1 g of K2HPO4, 0.5 g of MgSO4.7H2O, 0.5 g of KCl, 0.01 g of FeSO4, 30 g of glucose and 20 g of agar powder. Dilute them with deionized water to a total volume of 1000 ml, and sterilize them with high temperature steam at 121° C. for 30 min, so as to prepare a CDA medium. Aspergillus flavus strain is cultured with the CDA solid medium at 28° C. and 90% humidity for 10 days, and then the culture plate is washed with 20% Tween-20 to obtain a solution of Aspergillus flavus spores. Use the hemocytometer counting method, the Aspergillus flavus spore solution is shaken evenly with a vortex oscillator, and the Aspergillus flavus spore solution is counted microscopically with a microscope.
Put 15 mL of potato dextrose liquid medium in a 50 mL Erlenmeyer flask, autoclave the potato dextrose liquid medium at 121° C. for 30 min. Thereafter, based on the counting results, add the Aspergillus flavus spore solution until the final concentration is 1×105 spores per ml, and culture with shaking at 28° C. and 200 rpm for 96 h. Filter the culture broth with double-layer filter paper to obtain the filtrate (preserved for later use) and the fungus ball of Aspergillus flavus. For the fungus ball of Aspergillus flavus, squeeze the excess water with filter paper, and dry it in an oven at 65° C. for 12 hours to obtain dried bacteria. After cooling it to room temperature, store it at −70° C. for later use. The filtrate obtained through filtering is stored at 4° C. for later use.
The immunoaffinity purification-high performance liquid chromatography standard method is adopted to measure the concentration of aflatoxin in the filtrate (stored for later use as described above) after shaking culture of the spores of Aspergillus flavus, and then the conventional Nor-1 gene transcriptional quantity measuring method is adopted to measure the Nor-1 gene transcriptional quantity in the dried bacteria stored for later use.
Adopt the same operation as described above, seven Aspergillus flavus strains are cultured at different time periods before and after two batches are tested. The measuring results are shown in Table 1.
According to the measuring results in Table 1, there is a big difference between the concentration of aflatoxin in the two batches, and therefore the aflatoxin yield alone cannot serve to evaluate of the toxigenic capability of Aspergillus flavus strains. In some cases where the relative amount of Nor-1 gene transcription to Aspergillus flavus strains is high, the toxigenic capability is unexpectedly low. In addition, non-toxigenic strains can also transcribe the Nor-1 gene, and therefore the Nor-1 gene alone cannot serve to evaluate the toxigenic capability of Aspergillus flavus strains either. Surprisingly, in Table 1, in the ratio obtained by dividing the concentration of aflatoxin by the relative amount of Nor-1 gene transcription, that is, the [AFT]/[nor-1] value in Table 1, a high regularity is found. Not only that the orders of the toxigenic capability of the 7 strains obtained from the two culture batches are consistent, but also the [AFT]/[nor-1] value of each strain is relatively stable, and thus qualifying to evaluate the toxigenic capability of Aspergillus flavus strains.
It can be seen from the data in Table 1 that, if the ratio of the concentration of aflatoxin to the relative amount of Nor-1 gene transcription is adopted to evaluate the toxigenic capability of Aspergillus flavus strains, the accuracy and reliability of such as approach are significantly better than the approach of using the concentration of aflatoxin alone of the approach of using relative amount of Nor-1 gene transcription alone.
Utilize the known phage VHH 2-5 surface-displaying aflatoxin anti-idiotypic nano antibody and nor-1 gene DNA fragment Tq-nor1 to establish synchronous detection RT-PCR method for aflatoxin yield and Nor-1 gene transcriptional quantity. The above detection result is used as the scientific basis for identifying and evaluating the toxigenic capability of Aspergillus flavus strains, which provides support for the method of rapid identification and evaluation of the toxigenic capability of Aspergillus flavus strains. The specific steps are as follows.
The phage VHH 2-5 surface-displaying aflatoxin anti-idiotypic nano antibody is developed by the Quality Inspection Center of Oil Crop Research Institute, Chinese Academy of Agricultural Sciences, and has been published in the journal “Yanru Wang; Peiwu Li; Zuzana Majkova; Candace RS Bever; Hee Joo Kim; Qi Zhang; Julie E. Dechant; Shirley J. Gee; Bruce D. Hammock; Isolation of Alpaca Anti-Idiotypic Heavy-Chain Single-Domain Antibody for the Aflatoxin Immunoassay; Analytical Chemistry, 2013, 8298-8303”. The phage used in this example is stored in E. coli ER2738 in advance in the laboratory and obtained by amplification. The amplification method is as follows:
Pick a single colony randomly from the ER2738 single colony plate holding the nano antibody phage VHH 2-5, inoculate it into a liquid medium containing 1 mL of SB-ampicillin, and incubate overnight at 225 rpm in a constant temperature shaker at 37° C. Add the above overnight culture broth to 100 mL of SB-ampicillin liquid medium at 225 rpm and 37° C. and culture it to OD600=0.5-0.6. Add 1.5 ml of M13KO7 helper phage (titer 1×1011-1×1012 pfu/mL) to the cultured bacteria solution, and let it stand at 37° C. for 30 min. Add kanamycin with a final concentration of 70 μg/mL to the broth at 37° C. and 225 rpm, and shake culture it overnight. Centrifuge the broth cultured overnight at 4° C. and 10000 rpm for 15 minutes, take the supernatant, and transfer it to a clean centrifuge bottle. Add ¼ volume of PEG/NaCl and let it stand on ice for 2 hours. Centrifuge it at 10000 rpm for 30 minutes at 4° C., re-suspend it with 2 mL of 0.5% BSA/PBS, and then centrifuge it at 12000 rpm for 5 min. Take the supernatant, filter the supernatant with a 0.22 μtm filter and mix it with an equal volume of sterile glycerin, aliquot and measure the titer. The titer measuring method is as follows:
Randomly pick a single colony from the ER2738 single colony plate, inoculate it into a LB liquid medium containing 0.04 mg/mL of tetracycline, culture it overnight in a constant temperature shaker at 37° C. at 225 rpm. Take 20 μL of the above broth that is cultured overnight and add it to 2 mL SB medium. Incubate it at 225 rpm and 37° C. to OD600≈1. Use LB liquid medium to gradually dilute the phage that needs to be tested for titer. Take 10 μL of each dilution and add it to 100 μL of ER2738 broth with OD600≈1, and let it stand at 37° C. for 20 minutes. Make the phage infect E. coli; spread the infected E. coli broth on the LB-ampicillin plate, put it upright in a constant temperature incubator at 37° C. for 30 minutes, and then invert the culture overnight. Select about 100 of single colonies for plate counting, and calculate the titer of phage according to the following formula:
The method for obtaining the nor-1 gene DNA fragment Tq-nor1: Put 15 mL of potato dextrose liquid medium in a 50 mL Erlenmeyer flask, autoclave it at 121° C. for 30 min, and add the spore solution of No. N73 Aspergillus flavus until the final concentration is 1×105 ml −1. After shaking culture at 28° C., 200 rpm for 96 h, use double-layer filter paper to squeeze out the fungus ball culture solution, dried it at 65° C. for 12 h, quick-frozen it with liquid nitrogen, and ground it into powder. Accurately weigh 200 mg of hyphal powder, use the DNA extraction kit (DNeasy Plant Mini Kit) to extract the genomic DNA of strain N73 according to the kit instructions. Use the following primers to amplify the nor-1 gene fragment product with a size of 400 bp, and use E.Z.N.A.T.M (OMEGA) gel extract kit to purify the DNA fragment with a size of 400 bp according to the instructions to obtain Tq-nor1.
The DNA fragment Tq-nor1 of the DNA fragment and nor-1 gene released by the phage VHH 2-5 surface-displaying aflatoxin anti-idiotypic nano antibody is adopted as the template for RT-PCR synchronous amplification. Determine the ratio of aflatoxin yield to Nor-1 gene transcriptional quantity through synchronous RT-PCR amplification results.
Encoding the sequence of nano antibody and nor-1 gene sequence, the sequence of the upstream and downstream primers (Ph-F, Ph-R) of aflatoxin and the probe (Ph-probe), and the sequence of upstream and downstream primers (Tq-nor1-F, Tq-nor1-R) of Tq-nor-1 and the probe (Tq-probe) according to the phage VHH 2-5 surface-displaying aflatoxin anti-idiotypic nano antibody. Then use Oligo7.0 primer analysis software to perform verification according to the design principles and guidelines for primers and probes in the double RT-PCR reaction.
Primer and probe verification principle: The TM value of all primers should be set to the same or similar level, and the TM value of all probes should be as similar as possible and greater than the TM value of the primer by about 5° C. to 10° C. Because the reaction is a synchronous reaction of the same system, ensure that all primers and probes are not easily formed into dimers. Perform BLAST search to ensure that primers and probes are specific to the intended target.
It is determined that the following primers and probe sequences meet the above principles and requirements, as summarized in Table 2.
Synchronous RT-PCR reaction parameters are different from RT-PCR amplified through single gene, because it is necessary to carefully consider the interference between the reaction systems. First, RT-PCR amplify two single-stranded-specific DNA fragment of aflatoxin anti-idiotypic nanobody phage and DNA fragment of nor-1 gene. The reaction components are directly mixed without adding other components, and then perform double RT-PCR reaction, the result is shown in
For this reason, the disclosure optimizes the RT-PCR reaction conditions of the synchronous detection method for aflatoxin yield and Nor-1 gene transcriptional quantity. In the disclosure, the concentrations of DNA polymerase, MgCl2 and dNTPs are optimized. The result shown in
DNA polymerase: 0.5 U to 1.0 U; MgCl2: 1 mM to 2 mM; dNTPs: 200 uM to 400 uM.
According to the above research results, the disclosure provides the final optimized amplification reaction parameters, as shown in Table 3.
The synchronous RT-PCR amplification results of serially diluted phage and serially diluted nor-1 are shown in the synchronous RT-PCR amplification curve (RFU, relative fluorescent unit), as shown in
(1) Coating: Use PBS to dilute the commercially available aflatoxin monoclonal antibody 1C11 (secreted by hybridoma cell strain 1C11 with preservation number CCTCC NO: C201013, patent application number is CN201010245095.5, specific reports thereof are available) to 1.0 μg/mL. Use micropipette to add it to a 96-well microplate, 100 μL per well, incubate it overnight at 4° C. (≈12 h), wash the plate 3 times with PBST; block it: block each well with blocking solution, 300 μL per well, incubate it at 37° C. for 45 min, wash the plate 3 times with PB ST.
(2) Blocking: Block with blocking solution, 300 μL per well, incubate it at 37° C. for 45 min, wash the plate 3 times with PBST.
(3) Competition: Dilute the aflatoxin B1 standard product with 100% pure methanol to a concentration of 200 ng/mL, then dilute the standard solution of aflatoxin B1 with a 3-fold gradient of 10% (v/v) methanol/PBS, such that the concentration range is 33.33 ng/mL to 1.69 pg/mL. Then mix VHH 2-5 phage with known concentration (1.0×1010 cfu/mL) with aflatoxin B1 of 50 μL series concentration, add 10 μL of the mixture to 96-well microtiter plate. After incubation at 37° C. for 1 h, wash the plate with PBST for 10 times.
(4) Elution: Inject 90 μL of phage eluate into each well, leave it in a warm bath at 37° C. for 15 minutes, gently blow and hit it with a micropipette and transfer the eluate containing phagemid.
(5) Neutralization: Mix 90 μL of the removed solution with 10 μL of the neutralization solution to make the mixture neutral—the volume of the neutralization solution added is adjusted according to the actual pH value of the eluent and neutralization solution to ensure that the mixture is neutral.
In the immune reaction stage, under the condition that the coating amount of aflatoxin monoclonal antibody 1C11 is fixed, aflatoxin of different concentrations will compete with VHH 2-5 phage of different amounts to bind to 1C11. The greater the concentration of aflatoxin, the smaller the chance for phage to bind to 1C11 and the lower the binding amount. After the immune response is over, the phage bound to 1C11 will be eluted. The number of phages in the eluate is related to the concentration of aflatoxin. The phagemid in the eluate will release DNA molecules during the heating process of the PCR reaction. The released DNA molecules are used as the amplification target in the RT-PCR reaction. After the amplification reaction, the fluorescence quantification system software will provide the Ct values of amplification of phages of different amounts in different eluates corresponding to aflatoxin of different amounts. The Ct values obtained by serially diluting aflatoxin of different concentrations (33.33 ng/mL to 1.69 pg/mL) to the logarithm of aflatoxin concentration is subjected to four-parameter logistic regression by using Origin Pro 8.0 software, and the result is used as the S-type standard curve for the quantification of aflatoxin, as shown in
2. Establishment of Standard Curve for Quantifying nor-1 Gene Transcription
An Aspergillus flavus strain is taken. In this example, the Aspergillus flavus strain N73 preserved by the research center is used, which is a highly toxigenic strain and is used in the preparation of Tq-nor1 in this study. For other Aspergillus flavus strains, it is possible to use “method for obtaining DNA fragment Tq-nor1 of nor-1 gene” described above to amplify DNA fragment Tq-nor1 with a size of 400 bp. Both of them can be used as candidate strains for establishing the standard curve for quantifying nor-1 gene transcription. After using spectrophotometer (NanoDrop 2000, Thermo Scientific, U.S.A.) to detect the concentration of Tq-nor1, calculate the copy number of Tq-nor1. Tq-nor1 is serially diluted (102 to 108 copies) as a sample with known amount, and then subjected to RT-PCR amplification. Use Origin Pro 8.0 software, wherein the logarithm value of the Tq-nor1 standard sample serial copy number is the abscissa and the Ct value is the ordinate for performing regression analysis.
The standard curve for quantifying aflatoxin is shown in
Aflatoxin B2, G1, G2, Zearalenone (ZEN), Deoxynivalenol (DON), Fumonisin (FB1) standard products are diluted to a series of concentrations. After they compete with phage VHH 2-5 in the competitive immune reaction, the phage bound to the monoclonal antibody 1C11 at bottom of the microplate is eluted to undergo RT-PCR synchronous amplification with Tq-nor1. The Ct value obtained by the amplification corresponds to the logarithmic value of the different concentration of toxin to be used as the standard curve. Calculate the IC50 value, and calculate the cross-reactivity according to the cross-reactivity calculation formula % CR=(IC50 AFB1/IC50 analyte)×100. As shown in
Aflatoxin B1 standard product and DNA fragment Tq-nor-1 of nor-1 gene are simultaneously added to a 2 mL blank PDB medium. Shake and mix them well, and after mixing evenly, place the mixed solution at 4° C. and avoid light for 4 days. After 4 days, the mixture is diluted by 20 times, and the contents of aflatoxin B1 and Tq-nor-1 in the mixture are synchronously detected by RT-PCR. Set 3 replicates in the group of the same day, and set 3 replicates between groups of different days. The results of addition recovery are shown in Table 3:
The addition recovery rate of aflatoxin B1 is 88.37% to 103.10%, and the addition recovery rate of DNA fragment Tq-nor-1 of Nor-1 gene is 86.18% to 98.17%. This result shows that the established synchronous RT-PCR has reliable repeatability and reproducibility in actual sample detection and analysis.
5. Apply Synchronous RT-PCR Method to Quantify the Expression Level of Toxigenic Capability of Aspergillus flavus Strains and nor-1 Gene
In this study, 17 Aspergillus flavus strains with different toxigenic capability are selected. Put 15 mL potato dextrose liquid medium in a 50 mL Erlenmeyer flask, autoclave it at 121° C. for 30 min, add the Aspergillus flavus spore solution until the final concentration is 1×105 ml−1. After shaking culture at 28° C. and 200 rpm for 96 h, filter the culture broth with double-layer filter paper to obtain the strain culture broth and Aspergillus flavus fungus ball. Use filter paper to squeeze the excess water out of the Aspergillus flavus fungus ball and dry it for 12 hours at 65° C. by using an oven, cool it to room temperature, grind it into powder with liquid nitrogen, and accurately weigh 0.20 mg of fungus powder per sample. Follow the RNA extraction kit (RNeasy Plant Mini Kit) instructions for performing total RNA extraction. Then use QuantiTect reverse transcription kit to synthesize cDNA. The cDNA solution is diluted by 100 to 1000 times and replace Tq-nor1 in the synchronous RT-PCR amplification system, and used as one of the amplification templates to perform synchronous RT-PCR amplification to measure the nor-1 gene transcriptional quantity. After the strain culture medium is diluted by 10 times with 10% (w/v) BSA/PBS, it replaces the aflatoxin standard product in the immune reaction to participate in the immune competition reaction. After the competition reaction, the phagemid in the eluate is used as another template of the synchronous RT-PCR amplification system, and synchronous RT-PCR amplification is performed to measure the toxigenic capability of the strains. Use the synchronous RT-PCR method to quantify the toxin production and Nor-1 gene expression level of 17 Aspergillus flavus strains, and the results are as shown in Table 4.
aND
In order to verify the accuracy of the results, referring to the method of national standard GB5009.22-2016, a HPLC method is used to quantify the amount of toxin produced by Aspergillus flavus strains. Meanwhile, the gene expression of the strain nor-1 is quantified with a spectrophotometer (Nanodrop). The quantitative results are shown in Table 4. The result is compared with the result obtained through the synchronous RT-PCR method, and the comparison result is shown in
1. Use Nor-1 Transcriptional Quantity and AFT/Nor-1 (the Ratio of Toxigenic Capability to nor-1 Transcriptional Quantity) to Evaluate the Toxigenic Capability of Aspergillus flavus Respectively
Through the comparison and analysis of the results, it is found that if the toxigenic capability of Aspergillus flavus is evaluated solely from the nor-1 transcriptional quantity, the identification results are not reliable. For example, the logarithmic values of the copy number of nor-1 gene expression in Aspergillus flavus strains Pc124-2 and Pc34-1 are 7.85±0.52 and 6.75±0.58, respectively. The nor-1 gene expression levels of the two strains are equivalent. However, the production of aflatoxin of strain Pc124-2 is 66.5±4.93 ng/mL, and the production of aflatoxin of strain Pc34-1 is only 19.69±2.27 ng/mL. In addition, strains CY1, CY2, Pg28-1 and Pc321-1-3 are not found to produce aflatoxin. Nonetheless, the nor-1 gene levels expressed by strains Pg28-1 and Pc321-1-3 are even higher than those of some strains that produce aflatoxin.
However, it is more reliable to evaluate the toxigenic capability of Aspergillus flavus by using the ratio of production of aflatoxin to the expression level of nor-1 gene. In order to further explore the correlation relationship between the toxigenic capability of Aspergillus flavus strains, expression level of nor-1 gene as well as AFT/Nor-1, the toxigenic capability of strains is taken as the ordinate, and the logarithm of nor-1 gene expression amount and the ratio of AFT/Nor-1 are used as the abscissa for the diagram. The correlation relationship between toxigenic capability of Aspergillus flavus strains and Nor-1 gene expression amount is as shown in
In light of the above, the disclosure provides that the equation for identifying toxigenic capability of Aspergillus flavus strains is: y=10.14x−16.20, wherein X represents AFT/Nor-1, and Y represents toxigenic capability. For highly toxigenic strains, the toxigenic capability is >150 ng/mL; the medium toxigenic capability: 50<toxigenic capability<150 ng/mL; the low-toxigenic capability: toxigenic capability<50 ng/mL; no production of toxin: 0; then calculate the identification range of toxigenic capability according to the regression equation:
Highly toxigenic strains: AFT/Nor-1>16.4;
Medium toxigenic strains: 6.5<AFT/Nor-1<16.4;
Low toxigenic strains: 0<AFT/Nor-1<6.5;
Non-toxigenic strains: AFT/Nor-1=0.
Verification of Stability of AFT/Nor-1 (Ratio of Toxigenic Capability to nor-1 Transcriptional Quantity) in Identification Result of Toxigenic Capability
2. Verification of Stability of AFT/Nor-1 (Ratio of Toxigenic Capability to nor-1 Transcriptional Quantity) in Identification Result of Toxigenic Capability
In order to further verify the stability of AFT/Nor-1 ratio in identifying the results of toxigenic capability, intra-group and inter-group experimental analyses are respectively performed to five Aspergillus flavus strains in terms of toxigenic capability of aflatoxin and nor-1 gene transcriptional quantity. In the intra-group experiment, 5 replicates are set in parallel on the same day, and the average value of the 5 replicates is used as the toxigenic capability and nor-1 gene transcriptional quantity of the batch of strains. Three different dates are set for the inter-group experiment, each date is set as a batch, and a total of 3 batches are set.
The results are shown in Table 5.
#CV
&CV
1AFT
2AFT
3AFT
1Nor-1
2Nor-1
3Nor-1
1Ratio
2Ratio
3Ratio
@17.16
@24.17
@20.91
@33.23
@20.78
@42.00
@35.57
1AFT production of aflatoxin in the first batch;
2Nor-1 Nor-1 gene transcriptional quantity of the second batch;
3Ratio AFT/Nor-1 ratio of the third batch;
@The coefficient of variation (CV) is greater than 15%.
The amount of aflatoxigenic capability and Nor-1 gene transcriptional quantity in the table are the average of 5 repeated intra-group experiments.
From the table, it can be easily found that there is a big difference between the aflatoxin yield produced by different culture batches and nor-1 gene transcriptional quantity. Taking strain 233-1 as an example, the yields of aflatoxin detected in the three batches are 10.07, 19.69, and 25.32 ng/mL, respectively, and the coefficient of variation between the different batches is 42.00%. In addition, for the yields of aflatoxin produced by the strains IT-2, Pg56-1-2, N200 and 233-1, the intra-group coefficients of variation between different batches are all larger than 17.00%. It can be seen that it is unreliable to evaluate the toxigenic capability of strains from the aflatoxin yield produced alone. By analogy, based on the inter-group nor-1 gene transcriptional quantity between different batches, it can be found that there is a big difference between nor-1 gene transcriptional quantity, which equivalently proves that it is not reliable to identify and evaluate the toxigenic capability of strains solely from the nor-1 gene transcriptional quantity. However, the inter-group coefficients of variation of AFT/Nor-1 ratio are less than 13%, which has better stability.
In summary, the method for identifying and evaluating the toxigenic capability of toxigenic strains of aflatoxin provided in the disclosure, that is, the AFT/Nor-1 ratio identification, is a more accurate and reliable method for identifying and evaluating the toxigenic capability of Aspergillus flavus strains.
Obviously, the above-mentioned embodiments are merely examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is unnecessary and impossible to list all implementation methods here. Therefore, obvious changes or alterations of the disclosure still fall within the protection scope of the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811491692.9 | Dec 2018 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/121785 | 11/29/2019 | WO | 00 |
