Detection Of Aspergillus Flavus By The Helicase-dependent Isothermal DNA Amplification Technology

Objective：A new rapid method to detect Aspergillus flavus samples has beenestablished on the basis of thermostable Helicase-dependent isothermal DNAamplification(tHDA).A highly specific set of primers were synthesized to target theITS gene of Aspergillus fumigatus.Establishing facilitate and rapid detection ofAspergillus flavus by the tHDA to understand the advantages and disavantages ofamplification methods that can lay the foundation for the practical application ofexperimental study.Methods：Designing the sequence specific primers that suit for And usingprimer design software Primer is6.0for isothermal amplification system, the designof sequence specific primers by retrieving from the Ensembl Fungidatabase,comparing the Aspergillus ITS sequences, finding species-specific sequenceand using primer design software Primer is6.0. Extracting1×108spores ofAspergillus flavus by liquid nitrogen grinding method to Preparae the of DNAtemplate Aspergillus flavuswhich was fold diluted to be made from the templatelibrary.Nucleic acid was amplificated in vitro by BioHelix IsoAmp II of the tHDA kitproduced by BioHelix and the product yield is in a1.5%agarose gel electrophoresisand sequenced. Specificity and sensitivity were tested by comparing with PCRmethod.Results： According totheresultsofelectrophoresis and sequencing, itpreliminarily shows that this methodcan beused forthedetectionofAspergillus flavusby thereactionsystem and therequirementsestablishedbytheIsoAmp II tHDA kitkit. By comparisonwith other strains, detection of Aspergillus flavus bytHDAhasgoodspecificity.Compared withconventional PCR, Both can detect the concentration oftemplate DNA-fold diluted to200×10-6ng/mL，but according to the electrophoreticbands, the ordinary PCR bands brighter.Conclusion：（1）ThetHDAreaction systemestablished by IsoAmp II tHDA kitkitcan beused forthedetectionofAspergillus flavus, even Aspergillus genus,and it hasagood specificity（.2）Compared to ordinaryPCR, althoughthe sensitivity of tHDA wassimilar to the result of PCR method, but the electrophoretic bands is less obvious.（3）The principle and operation ofthetHDAdetection methodisrelatively simple. It doesnot need specialinstruments,complex primers and higher operating conditionsandithas more advantagescomparingwithotherdetection techniques.But the followingaspects such as how to effectivelyextractthesampleof Aspergillus flavusgenome, howtosimplifythedetection step, how to avoid false positive results, and how to improveitssensitivityis need for furtherresearch.