Development Of Simultaneous Detection Method For Agro-food Born Fungi And Mycotoxin

Mycotoxins,poisonous secondary metabolites of fungi(Aspergillus,Fusarium,and Penicillium),can induce acute and chronic toxic effects on animals and humans,which represent a severe threat to food safety and security.To improve the safety level of food and feedstuffs,timely,and simultaneously,mycotoxigenic fungi detection,along with produced toxins,is very critical.Nowadays,Simultaneous detection technologies of mycotoxigenic fungi and related toxins has been becoming a hot topic and tendency in food safety and security to increase monitoring efficiency.Hence,many mycotoxigenic fungi detection technologies have been reported to measure and control mycotoxins contamination in food and feed substrates.In this study,the simultaneous detection method of mycotoxigenic fungi belonging to three different fungal genera,and Ochratoxin A and its producing fungi(Aspergillus ochraceus)were developed.Firstly,a multiplex polymerase chain reaction assay was developed for the simultaneous detection of mycotoxigenic fungi belong to Aspergillus,Fusarium,and Penicillium genera.In the present study,the developed multiplex polymerase chain reaction assay would allow a rapid,specific,and simultaneous detection of various mycotoxigenic potential fungi based on the present and size of the amplification products.In this study,three pairs of genus-specific primers were designed based on the internal transcribed spacer(ITS)sequences of Aspergillus and Penicillium,and Elongation factor 1 alpha(EF1?)of Fusarium.And the amplicons of 170,750,and 490bp for the corresponding primer pairs were detected,respectively.The sensitivity of the developed method was tested with genomic DNA obtained from pure mould cultures and artificially contaminated maize grain powder.And the result of the sensitivity showed that a spore concentration of 102spores/mL was detected from the contaminated maize grain powder without prior incubation.This result suggests that the developed mPCR assay would allow a rapid,specific,and simultaneous detection of various mycotoxigenic potential fungi based on the occurrence and size of the amplification products and thus to estimate the multimycotoxins contamination potential in food and feedstuff.Secondly,a duplex competitive immune real-time PCR assay was developed for simultaneous detection of Ochratoxin A,and its producing fungi,A.ochraceus,based on the advantages of the unique characteristics of VHH-2-24 phages.Ochratoxin A is detected based on the VHH-2-24 phages containing Ochratoxin A anti-idiotypic nanobody and its encoding DNA,which was used to design the specific primers,while for A.ochraceus,Ochratoxin A-synthesis related gene Pks was used.The optimized primers and probes system(0.25?M)for competitive immune-real-time-PCR assay were selected as optimal working concentration.Before duplex real-time PCR assay development,singleplex real-time PCR assays were developed for VHH-2-24 phages,and Pks-gene.The single-plex realtime PCR assays showed that Cq mean values were obtained when the concentrations of VHH-2-24 phages(pfu/mL),and Pks-gene(copies/?L)were as low as 101pfu/mL,and high as 109copies/?L.The plot of the Cq mean values against the log number of VHH-2-24 phages,and Pks-gene showed a good corrclation with an R2 value of 0.970,and 0.988 respectively.The amplification efficiencies of VHH-224 phages,and Pks-gene were calculated as 109%,and 90%,respectively,showing that the single-plex real-time PCR can amplify the VHH-2-24 phages and Pks-gene separately with the lowest detectable concentration of 101.These two single-plex real-time PCR assays were combined into duplex real-time PCR assay to detect VHH-2-24 phages,and Pks-gene simultaneously.Cq mean values were obtained simultaneously for both VHH-2-24 phage particles(pfu/mL),and Pks-gene DNA(copies/?L)with low(101),and high(109)concentrations;in other words,we can say that the duplex real-time PCR can detect VHH-2-24 phage particles and Pks-gene DNA from 101 to 109copies/?L concentrations simultaneously.The plot of the Cq mean values against the log number of VHH-2-24 phage,and Pksgene DNA showed a good correlation with an R2 value of 0.9949,and 0.969 respectively.The efficiency(E)of the duplex-real-time PCR was calculated in a good range of 91.5%,and 110%for VHH-2-24 phage particles,and Pks-gene DNA,respectively.Overall the analytical technique developed here for simultaneous detection of A.ochraceus and OTA,provides a platform for the advancement of new ideas about simultaneous detection technologies.