A novel Loop-mediated Isothermal Amplification Method for Screening Detection of Genetically Modified Cotton

BACKGROUND: Limited domestic cotton production in Japan has led to large amounts of cottonseed being imported for food and feed. Consequently, genetically modified (GM) cotton seeds have been introduced into the country, and a qualitative method is required for the detection of unauthorized GM cotton. OBJECTIVE: The aim of this study is to develop a testing method that is simpler, more rapid, and more cost-effective than conventional polymerase chain reaction (PCR) technique. METHODS: A novel method was developed for the screening detection of unauthorized GM cotton seeds using loop-mediated isothermal amplification (LAMP) to target the 35S promoter of the cauliflower mosaic virus (P35S), neomycin phosphotransferase Ⅱ (NPTII), and the endogenous cotton gene stearoyl acyl carrier protein desaturase (sad1). RESULTS: The method limit of detection (LOD) was determined to be ≤ 0.1%, which is equivalent to the corresponding LOD achieved using the conventional PCR method. A simpler detection method based on single-stranded tag hybridization on a chromatography-printed-array strip system (denoted DNA chromatography) was also developed. This method allowed visual detection of the amplification of multiple DNA samples at the LOD level. CONCLUSIONS: Compared with the conventional PCR approach, the developed detection method is cost-effective, rapid, and simple. HIGHLIGHTS: The developed method facilitates the analysis of GM crops and will be expected to help prevent the unintended spread of GM crops into the environment.