| CPC C12Q 1/6895 (2013.01) [C12Q 1/686 (2013.01); C12Q 2600/158 (2013.01)] | 4 Claims |
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1. A method for identifying and evaluating toxigenic capability of aflatoxigenic strain, comprising:
culturing Aspergillus flavus strains to obtain an Aspergillus flavus spores solution; culturing the Aspergillus flavus spores solution; and then filtering to obtain a strain culture solution containing aflatoxin and a fungus ball after the shaking culture is completed;
measuring an aflatoxin yield of the strain culture solution and a Nor-1 gene transcriptional quantity of the fungus ball to obtain a ratio of the aflatoxin yield to the Nor-1 gene transcriptional quantity; and
identifying and evaluating the toxigenic capability of the aflatoxigenic strain according to the ratio of the aflatoxin yield to the Nor-1 gene transcriptional quantity,
wherein when the ratio is greater than 16.4, it indicates that the aflatoxigenic strain is a highly toxigenic strain having a toxigenic capability greater than 150 ng/mL; when the ratio is greater than 6.5 and less than 16.4, it indicates that the aflatoxigenic strain is a medium toxigenic strain having a toxigenic capability greater than 50 ng/ml and less than 150 ng/ml; when the ratio is greater than 0 and less than 6.5, it indicates that the aflatoxigenic strain is a low toxigenic strain having a toxigenic capability less than 50 ng/mL; when the ratio is 0, it indicates that the aflatoxigenic strain is a non-toxigenic strain,
wherein the aflatoxin yield and the Nor-1 gene transcriptional quantity is measured through a synchronous detection RT-PCR method, and the synchronous detection RT-PCR method comprises the following steps:
(1) establishing an S-type standard curve for quantifying aflatoxin by the following steps: performing an immune competition reaction using aflatoxin standard products of different concentrations, a phage having surface-displaying aflatoxin anti-idiotypic nano antibody and an aflatoxin monoclonal antibody of constant amount, wherein the aflatoxin standard products compete with the phage to bind to the aflatoxin monoclonal antibody; eluting the phage bound to the aflatoxin monoclonal antibody to obtain eluates after the immune competition reaction is over, wherein the aflatoxin standard products of different concentrations correspond to different amounts of eluted phage in the eluates; performing a synchronous RT-PCR amplification reaction to obtain different first Ct values using DNA molecules of the eluted phage as amplification targets in each of the eluates separately; and performing a regression analysis to obtain the S-type standard curve using a logarithm of the different concentrations of the aflatoxin standard products as abscissa and the different first Ct value as ordinate;
(2) establishing a RT-PCR standard curve for Nor-1 gene transcriptional quantity by the following steps: serially diluting samples of known copy number of Nor-1 gene DNA fragment (Tq-nor1) into different copy numbers; performing the synchronous RT-PCR amplification reaction to obtain different second Ct values using each copy number of the Tq-nor1 separately; and performing a regression analysis to obtain the RT-PCR standard curve using a logarithm of the different copy number of the Tq-nor1 as abscissa and the different second Ct value as ordinate;
(3) diluting the strain culture solution containing the aflatoxin; performing another immune competition reaction using the diluted strain culture solution, a phage having surface-displaying aflatoxin anti-idiotypic nano antibody and aflatoxin monoclonal antibody of constant amount, wherein the aflatoxin competes with the phage to bind to the aflatoxin monoclonal antibody; eluting the phage bound to the aflatoxin monoclonal antibody to obtain eluate after the immune competition reaction is over; performing a synchronous RT-PCR amplification reaction to obtain a third Ct value using DNA molecule of the eluted phage as amplification template; additionally, drying the fungus ball, extracting a total RNA from the dried fungus ball, and reverse transcribing the total RNA into cDNA; and performing synchronous RT-PCR amplification reaction to obtain a fourth Ct value using the cDNA as amplification template; calculating the aflatoxin yield corresponding to the third Ct value using the S-type standard curve; and calculating Nor-1 gene transcriptional quantity corresponding to the fourth Ct value using the RT-PCR standard curve;
wherein in the reaction system of the synchronous RT-PCR amplification reaction, primers used to amplify Nor-1 gene DNA fragment (Tq-nor1) are as follows:
upstream primer Tq-nor1-F, wherein the nucleotide sequence of the upstream primer Tq-nor1-F is SEQ ID NO: 4; and
downstream primer Tq-nor1-R, wherein the nucleotide sequence of the downstream primer Tq-nor1-R is SEQ ID NO: 5;
wherein in the reaction system of the synchronous RT-PCR amplification reaction, primers used to amplify the DNA molecules released by the phage surface-displaying aflatoxin anti-idiotypic nano antibody are as follows:
upstream primer Ph-F, wherein the nucleotide sequence of the upstream primer Ph-F is SEQ ID NO: 1; and
downstream primer Ph-R, wherein the nucleotide sequence of the downstream primer Ph-R is SEQ ID NO: 2.
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