| Edible oil is an important source of essential fatty acids and energy the human body requires.Due to the growing demand for edible oil in domestic and international markets,the adulteration of low-priced oil with high-priced oil has become a problem that violates consumer rights and affects and damages the development of the industry,and there is an urgent need for reliable testing methods to solve such problems in the market.Traditional physical,chemical and spectroscopic methods are difficult to provide reliable results for detecting different varieties of oils.DNA-based detection methods can effectively avoid the defects of traditional detection methods that cause changes in fatty acids and other components in the used vegetable oils due to changes in external factors,and DNA has high genetic stability.Different oil plants contain different specific DNA.Ladder-shape melting temperature isothermal amplification(LMTIA)is a novel nucleic acid isothermal amplification technique with the advantages of high specificity,high sensitivity and simple operation,which has been used in the detection of African swine fever,adulteration detection of meat products,foodborne pathogenic bacteria It has been used in the detection of African swine fever,adulteration of meat products and foodborne pathogens.In this paper,based on the isothermal amplification technique of ladder melting temperature,we study the application of this technique in the DNA detection of edible oil as follows.1.To investigate the effect of target sequence selection on LMTIA detection.The following conclusions were obtained: the system using the transcribed spacer region as the target was more sensitive,the optimum temperature was 59℃,the minimum detection limit was 1 pg/μL.The system using thaumatin-like protein as the target had an optimal reaction temperature of 58℃ and a minimum detection limit of 100 pg/μL.Good specificity for both systems,no false positives for either system.2.To prepare LMTIA positive clone plasmid.The following conclusions were drawn:the sequencing results were compared with the software DNAMAN and the Chinese plant DNA barcode database.The duplicate sequences could reach 100% without mutation,which could be used as a positive control for subsequent experiments.3.To establish a soybean-derived LMTIA detection system.The enzymatic probe(Proofman)was also added to enhance the specificity and sensitivity of the reaction.The following conclusions were obtained: the optimum temperature was 57℃,the minimum detection limit was 1 pg/μL,and the specificity and stability were good.4.To test the suitability of the LMTIA system and edible oil DNA extraction,11 edible oil DNA extraction methods were collected in this experiment.The DNA was extracted from laboratory self-pressed soybean oil,supermarket-purchased refined first grade soybean oil and pressed first grade corn germ oil.Then,an ultra-micro nucleic acid protein analyzer determined the concentration and purity,followed by the LMTIA assay and fluorescent PCR method for validation.The following conclusions were drawn: these 11 edible oil DNA extraction methods can be divided into three categories in terms of their working principles,namely emulsification,magnetic bead method,and centrifugal column method.According to the DNA extraction efficiency,magnetic bead method > emulsification method > centrifugal column method.From the LMTIA test results,SOP magnetic bead method can extract amplifiable DNA for all three kinds of edible oils,and it is the best for the extraction of laboratory self-pressed soybean oil.The extraction success rate is not high for refined oil.The hexane emulsification method could also extract amplifiable DNA.Still,it was mostly fragmented and not high purity,and the efficiency and success rate were not increased from the amplification results.Amplification of extracted soybean oil DNA by commercially available fluorescent PCR method amplifies only laboratory self-extracted soybean oil DNA. |
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