Research On Detection Method Of Mold In Nuts Food

Mold is the common name for filamentous fungi,mold contamination is the main reason for unqualified quality inspection of nuts,and the rapid detection of mold contamination is of great significance to the development of the nut industry.The current national standard method needs 5 days for mold detection,which seriously restricts the rapid development of the industry.Aspergillus,Penicillium and Fusarium belong to the nut mould contamination is the main pollution.In recent years,the rapid development of molecular biology technology and time-resolved fluorescence analysis technology has provided an important basis and innovative means for food safety detection.However,the application in the field of mold detection is still less.In this study,several nanomaterials were prepared by hydrothermal method and other methods.A series of rapid,sensitive and simple detection methods were constructed by combining molecular beacon-q PCR technology,fluorescence resonance energy transfer technology and loop-mediated isothermal amplification（LAMP）.Firstly,according to the internal transcribed spacer gene sequences of Aspergillus,Penicillium and Fusarium,primers and three pairs of specific molecular beacon probes were designed to establish a multiplex real-time fluorescence quantitative PCR for the simultaneous detection of Aspergillus,Penicillium and Fusarium in nuts.The q PCR reaction system was optimized,and good amplification curve and standard curve were determined.The results show that the minimum detection limit of Aspergillus and Penicillium were2.5×10^-2 ng/m L（741 CFU/g）and 8.7×10^-3 ng/m L（500 CFU/g）respectively.The minimum detection of Fusarium was 5.6×10^-3 ng/m L（454 CFU/g）,which provided a rapid and accurate method for the detection of mold in nuts.Compared with the national standard test method,it takes 5 days,while the method established in this research only takes 3 days.Secondly,two different types of KYF₄:Ln³⁺（Ln=Tb,Eu）nanoparticles with time-resolved fluorescence characteristics（TRNPs）and GO/Fe₃O₄ magnetic materials with highly efficient fluorescence quenching were synthesized.By modifying TRNPs molecular beacons of Aspergillus and Penicillium to form fluorescence probes,the two-color time-resolved fluorescence materials.By optimizing the experimental conditions,the linear range of Aspergillus gene detection was 2.5 nmol/L-50 nmol/L（y=106.24x-80.07,R²=0.9367）and the detection limit was 0.91 nmol/L（477 CFU/g）.The linear range of Penicillium gene detection was 2.5 nmol/L-50 nmol/L（y=160.46x-203.49,R²=0.9940）,and the limit detection was 1.21 nmol/L（634 CFU/g）.Finally,the FITC@Au NCs nanomaterial with double emission fluorescence was synthesized.Three pairs of primers were designed according to the 28S r RNA gene sequence of Aspergillus,and a ratio fluorescence biosensor based on gold nanoclusters combined with LAMP was established for the determination of Aspergillus.Bovine serum albumin（BSA）-protected Au NCs were used as reference fluorescence signal,and luciferin-isothiocyanate（FITC）was used as p H response signal.When there was a target nucleic acid in the system,LAMP reaction resulted in a decrease in p H in the system,resulting in a linear range of proportional fluorescence for Aspergillus gene detection ranging from 10^-3 to 10³ ng/m L（y=-0.12x+5.15,R²=0.9696）,and the limit detection was 7.4×10^-3ng/m L（481 CFU/g）.