Development Of Rapid Detection Methods Of Aspergillus Flavus And Aflatoxin B₁ In Food

Aflatoxins are highly toxic and carcinogenic secondary metabolism produced by fungi such as Aspergillus flavus（A.flavus）.Aflatoxins are common pollutant in grain and its products,which brings a great threat to food safety.Therefore,early detection of A.flavus and aflatoxins contaminated food helps to control aflatoxins.However,the traditional detection methods have some shortcomings,such as complex operation,high technical and equipment requirements,which cannot meet the needs of rapid detection.Therefore,we developed the rapid detection methods of A.flavus and AFB₁ in food.The main results of this subject are as follows:1.A real-time PCR method for the detection of A.flavus in grains was developed.PCR primers were designed for the Internal Transcribed Spacer（ITS）and aflatoxins synthesis genes（including afl R,afl D and afl P）of A.flavus,and the“ITSⅠ”primers were selected for the following assay.The specificity and sensitivity of the method were evaluated,and the results showed that it had high specificity and could detect most types of A.flavus.The sensitivity of it was 10⁶ conidia/g,which is equal to the reported method using FLAQ primers.This method can be used for daily detection of A.flavus.2.An on-spot and rapid recombinase polymerase amplification（RPA）assay for A.flavus detection in grains was developed.A method for rapid extraction of DNA from grains by washing,filtration and grinding operations was established.Among this method,0.5 M sodium hydroxide（Na OH）was used as grinding buffer,and simple tools were made.The method can extract DNA in 10 min.Three RPA primer pairs were designed,and then a suitable primer pair was selected.The RPA reaction system was optimized so that it could be completed by 10 min at 37℃.At the same time,Celfinder^TM nucleic acid dye was selected for visual detection.The established RPA assay can achieve high sensitivity detection of A.flavus in grains in 30 min,and its sensitivity can reach 10¹ conidia/g.Its practical application has also been verified.Meanwhile,the whole detection process does not need expensive equipment,it is very suitable for on-spot detection of A.flavus,which is of positive significance to control the spread of A.flavus.3.An aptasensor model based on CRISPR-Cas14a for AFB₁ detection was designed and developed.First,the single-strand DNA（ss DNA）detection system based on CRISPR-Cas14a was established and optimized.It can achieve high sensitivity detection of ss DNA in a short time,and the LOD was as low as 0.5 n M.Secondly,the feasibility of the aptasensor model was explored.The aptasensor can produce signal response in the presence of AFB₁,although the sensitivity of the sensor was still low after optimization and it can only be used for the detection of samples with AFB₁ content above 100 ng/m L,the detection model combined CRISPR-Cas14a system with aptamer for the first time and expands the detection range of CRISPR from nucleic acid target to small molecule target,which was of positive significance to the development of new detection methods.At the same time,the detection sensitivity can be improved by optimizing aptamers and their complementary sequences in the future.