Detection of loop-mediated isothermal amplification based on Nicotiana-specific gene Ntsp151.

The 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 tobacco materials 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 showed 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 amplification temperature 63 °C, reaction time 60 min, Mg2+ concentration 6 mmol/L; LAMP detection method had good specificity and the results of chromogenic amplification and fluorescence detection of 17 non-tobacco materials and 1 tobacco material were consistent and had good specificity. The sensitivity test showed that the minimum detection limit of the LAMP reaction system was 102 copies/μL. The LAMP detection based on Nicotiana-specific gene Ntsp151 was result-visualized, highly sensitive, and specific, which was suitable for field inspection. [ABSTRACT FROM AUTHOR]