Development Of Nucleic Acid Amplification Based Detection Method For Ochratoxin A In Agricultural Products

Ochratoxins,a serial of secondary fungal metabolites,was mainly produced by several Aspergillus species and Penicillium species.Ochratoxin A?OTA?was characterized by a large polluted area and deleterious effects.And it hard to remove OTA from agro-products.Thus,it's much important to develop a detection method with high sensitivity.In recent years,the nucleic acid amplification was extended to the detection of mycotoxins and was also regarded as an important platform to help the simultaneous detection of both mycotoxin and food-borne pathogens.However,because mycotoxins were small molecular without DNA sequence,it was hard to make a breakthrough to apply the nucleic acid amplification into the mycotoxin detection.In order to deal with the problems above,this research employed the phage of anti-idiotypic nanobody of OTA?phage VHH 2-21?as material,which was characterized by possessing both antibody and DNA sequence,to develop three kinds of real-time immune PCR?real-time IPCR?approaches for OTA detection.The main contents and innovations of this research are as follows1.The development of a real-time IPCR method based on the nucleic acid dye of SYBR Green?for OTA detection.In this research,the phage VHH 2-21 was employed as research material.The optimal working concentration of phage VHH 2-21 and 1H2 mAb were obtained by the checkerboard method.The specific primer was designed according to the DNA sequence of phage VHH 2-21.The phage VHH 2-21 of known concentration was employed to optimize the amplification conditions of the SYBR Green?based real-time IPCR method.Under the optimal conditions,the standard curve of the SYBR Green?based real-time IPCR method was established.The IC₅₀ of this real-time IPCR was 0.03 ng/mL with the limit of detection?LOD?of 0.003 ng/mL.Corn,rice,and wheat were used as three kinds of matrixes to evaluate the matrix effect of the SYBR Green?based real-time IPCR method.Corn matrix was dealt with by petroleum ether to remove oil.And BSA was added into all kinds of matrix solutions.The conclusion was there was little matrix effect in this real-time IPCR method.The recovery analysis was conducted with the concentration of the OTA standard solution ranged from 2 to 10 ng/mL.The average recovery was ranging from 88.7%-113.4%,which satisfied the common request of recovery?80%-120%?.Thus,the SYBR Green?based real-time IPCR method suited for OTA detection in agro-products.2.The development of a real-time IPCR detection method based on the Taqman probe for OTA detection.In this research,the phage VHH 2-21 was employed as research material.The specific primer and Taqman probe were designed according to the DNA sequence of phage VHH 2-21.The phage VHH 2-21of known concentration was employed to optimize the amplification conditions of the Taqman probe-based real-time IPCR method.Under the optimal conditions,the standard curve of the Taqman probe-based real-time IPCR method was established.The IC₅₀ of this real-time IPCR was 0.012 ng/mL with the LOD of 0.001 ng/mL.Corn,rice,and wheat were used as three kinds of matrixes to evaluate the matrix effect of the Taqman probe-based real-time IPCR method.Corn matrix was dealt with by petroleum ether to remove oil.And BSA was added into all kinds of matrix solutions.The conclusion was there was little matrix effect in this real-time IPCR method.The recovery analysis was conducted with the concentration of the OTA standard solution ranged from 2 to 10 ng/mL.The average recovery was ranging from 88.3%-109.6%,which satisfied the common request of recovery?80%-120%?.Thus,the Taqman probe-based real-time IPCR method suited for OTA detection in agro-products.3.The development of a real-time multiply-primed rolling circle amplification?RCA?method for OTA detection.In this research,the phage VHH 2-21 was employed as research material.The phage VHH 2-21 of known concentration was employed to optimize the amplification conditions of the real-time multiply-primed RCA method.Under the optimal conditions,the standard curve of the real-time multiply-primed RCA method was established.The IC50 of the real-time multiply-primed RCA was 0.044 ng/mL with the limit of detection?LOD?of 0.004 ng/mL.Corn,rice,and wheat were used as three kinds of matrixes to evaluate the matrix effect of the real-time multiply-primed RCA method.Corn matrix was dealt with by petroleum ether to remove oil.And BSA was added into all kinds of matrix solutions.The conclusion was there was little matrix effect in the real-time multiply-primed RCA method.The recovery analysis was conducted with the concentration of the OTA standard solution ranged from 2 to 10 ng/mL.The average recovery was ranging from 88.2%-112.2%,which satisfied the common request of recovery?80%-120%?.Thus,the real-time multiply-primed RCA method suited for OTA detection in agro-products.