Establishment and Application of Loop-Mediated Isothermal Amplification for Tea-Cigarette Identification.

To provide a rapid and simple test method for tea-cigarette identification, a loop-mediated isothermal amplification (LAMP) primers were designed for the conserved region of the selected Nicotiana-specific gene Ntsp151 sequence, and a visual LAMP detection method for tea-cigarette based on the SYBR Green I was established. The genomic DNA extraction method and reaction conditions were optimized in the detection process. Furthermore, the specificity and sensitivity of the LAMP reaction system were evaluated. The results exhibited that the genomic DNA extracted by Chelex-100 could be directly amplified and fluorescently colored by LAMP; the optimal reaction conditions of the LAMP primers were the amplification temperature 63 ℃, reaction time 60 min, Mg2+ concentration 6 mmol/L; LAMP detection method had good specificity and the results of the chromogenic amplification and fluorescence detection of 17 non-tobacco materials and 1 tobacco material were consistent. The sensitivity test showed that the minimum detection limit of the LAMP reaction system was 102 copies μL-1. The detection of LAMP based on Nicotiana-specific gene Ntsp151 was visualized, highly sensitive and specific, which was suitable for field inspection.