In recent years,the prevalence of food allergies has steadily risen.For rapid detection of soybean and wheat allergen components in allergenic products,this study aimed to establish a rapid analysis method based on la...In recent years,the prevalence of food allergies has steadily risen.For rapid detection of soybean and wheat allergen components in allergenic products,this study aimed to establish a rapid analysis method based on ladder-shape melting temperature isothermal amplification(LMTIA)combined with proofreading enzyme-mediated probe cleavage(Proofman).The specific primers together with the Proofman Probe were to be designed for the soybean Lectin gene and wheat GAG56D gene sequences.The temperature of the duplex Proofman-LMTIA reaction was to be optimized,the specificity was to be determined,the sensitivity and detection limit were to be tested,and the efficacy of the duplex Proofman-LMTIA method was to be assessed with the commercial products as well as by comparison with the standard LAMP method.The duplex Proofman-LMTIA method achieved the amplification within 30 min at an optimal temperature of 62℃,the specificity had been validated by five other species,the sensitivity was 10 pg/μL soybean or wheat genomic DNA,and the detection limit was 5%of the allergenic soybean(or wheat)in non-allergenic soybean(or wheat).In addition,the results of nine types of commodities tested with the established duplex Proofman-LMTIA method were consistent with the allergy labels on the products,while the standard LAMP method had the false-positive results.The LMTIA approach can be effective for the rapid and precise identification of soybean and wheat allergen components in allergenic goods,and the practical application of this method will facilitate the market supervision.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.32172300)the Technology Project of Henan Province(Grant No.242102321128)+1 种基金the Central Government Guides the Local Science and Technology Development Special Fund(Nos:Z20231811102)as well as Henan Province University Science and Technology Innovation Talent Project(Grant No.23HASTIT048).
文摘In recent years,the prevalence of food allergies has steadily risen.For rapid detection of soybean and wheat allergen components in allergenic products,this study aimed to establish a rapid analysis method based on ladder-shape melting temperature isothermal amplification(LMTIA)combined with proofreading enzyme-mediated probe cleavage(Proofman).The specific primers together with the Proofman Probe were to be designed for the soybean Lectin gene and wheat GAG56D gene sequences.The temperature of the duplex Proofman-LMTIA reaction was to be optimized,the specificity was to be determined,the sensitivity and detection limit were to be tested,and the efficacy of the duplex Proofman-LMTIA method was to be assessed with the commercial products as well as by comparison with the standard LAMP method.The duplex Proofman-LMTIA method achieved the amplification within 30 min at an optimal temperature of 62℃,the specificity had been validated by five other species,the sensitivity was 10 pg/μL soybean or wheat genomic DNA,and the detection limit was 5%of the allergenic soybean(or wheat)in non-allergenic soybean(or wheat).In addition,the results of nine types of commodities tested with the established duplex Proofman-LMTIA method were consistent with the allergy labels on the products,while the standard LAMP method had the false-positive results.The LMTIA approach can be effective for the rapid and precise identification of soybean and wheat allergen components in allergenic goods,and the practical application of this method will facilitate the market supervision.
