Rapid detection of wheat components in starch products using the Proofman-LMTIA method

For rapid detection of wheat components in starch products, this study developed the analysis method based on the ladder-shape melting temperature isothermal amplification (LMTIA) coupled with proofreading enzyme-mediated probe cleavage (Proofman). With the internal transcribed spacer (ITS) gene of Triticum aestivum L. (wheat) as the target, specific primers and the Proofman probe were designed, the reaction temperature was optimized, and the sensitivity and the specificity of the Proofman-LMTIA method were determined. With the results indicating that the optimal reaction temperature of this Proofman-LMTIA method was 65°C, it can specifically detect wheat components rather than seven other species; the sensitivity was 10 fg (femtograms) of the genomic deoxyribonucleic acids (gDNAs) of Triticum aestivum L. and the detection limit was 0.1% of the artificially adulterated wheat starch. The Proofman-LMTIA reaction could be completed in 20 minutes, and 1 of 12 commercial starch products was found to have adulterated wheat starch. In conclusion, the established Proofman-LMTIA method was suitable, or rapid and accurate, for the detection of wheat components in starch products.